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MEDICAL BIOCHEMISTRY
This page intentionally left blank
 MEDICAL
BIOCHEMISTRY
SECOND EDITION
ANTONIO BLANCO†
Emeritus Professor, National University of Cordoba, Argentina

GUSTAVO BLANCO, M.D. AND PH.D.

Professor, Kansas University Medical Center, University of Cordoba, Argentina

†
Deceased
Academic Press is an imprint of Elsevier
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Copyright © 2022 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
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This book and the individual contributions contained in it are protected under copyright by the Publisher (other
than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our
understanding, changes in research methods, professional practices, or medical treatment may become
necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using
any information, methods, compounds, or experiments described herein. In using such information or methods
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from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
ISBN 978-0-323-91599-1

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visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci

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Typeset by STRAIVE, India
 Dedication

To the memory of my father

GB
This page intentionally left blank
 Contents

Preface xi Globin 53
Hemoglobin derivatives 58
Introduction xiii Abnormal hemoglobins 59
Blood plasma proteins 61
1. Chemical composition of living beings Main plasma proteins 62
Biogenic elements 1 Muscle proteins 67
Biological compounds 2 Muscle contraction 70
Summary 3 Proteomics 72
Further reading 3 Summary 72
Further reading 75

2. Water 4. Carbohydrates
The water molecule is polar 6 Monosaccharides 77
Hydrogen bond 6 Disaccharides 87
Water as solvent 7 Polysaccharides 88
Water as an electrolyte 8 Summary 101
Equilibrium constant 9 Further reading 103
Equilibrium of water ionization 10
Acids and bases 11
5. Lipids
The strength of acids and bases 12
pH 12 Classification 105
Buffers 13 Fatty acids 105
Titration curve of acids and bases 15 Essential fatty acids 110
Appendix 16 Simple lipids 110
Summary 18 Complex lipids 114
Further reading 19 Substances associated with lipids 121
Summary 127
3. Proteins Further reading 129

Proteins are macromolecules formed by amino acids 21 6. Nucleic acids

Amino acids 22
Peptides 31 Nucleotides 131
Proteins 33 Nucleic acids 133
Collagen 49 Virus 145
Keratins 51 Free nucleotides 148
Hemoglobin 52 Summary 150
Heme 52 Further reading 152

vii
viii Contents

7. Elements of thermodynamics and 11. Membranes

biochemical kinetics
Structure 233
Thermodynamics 153 Transport across membranes 242
The cell as an open system 158 Passive transport or facilitated diffusion 244
Chemical kinetics 160 Active transport 254
Summary 163 Summary 276
Further reading 164 Further reading 278

8. Enzymes 12. Digestion—Absorption

Enzymes are biological catalysts 165 Saliva 281
Nomenclature and classification of enzymes 165 Gastric secretion 284
Chemical nature of enzymes 170 Pancreatic juice 288
Metalloenzymes 171 Intestinal mucosa 291
Enzymatic catalysis 171 Bile 292
Active site 172 Summary of the digestive process 296
Zymogens 173 Absorption 299
Genetic alterations that affect enzyme function 174 Gastrointestinal microbiome 304
Enzyme distribution within the cell 174 Summary 305
Multienzyme systems 175 Further reading 306
Determination of enzyme activity 175
Factors that modify enzyme activity 175 13. Metabolism
Enzyme inhibitors 179
Regulation of enzyme activity 183 Metabolic pathways 307
Constitutive and inducible enzymes 185 Metabolic studies 309
Enzymatic processes in cascade 186 Regulation of metabolism 312
Isozymes 186 Summary 313
Determination of enzymes in the clinical laboratory 187 Further reading 314
Summary 189
Further reading 190 14. Carbohydrate metabolism
9. Biological oxidations: Bioenergetics Glucose uptake into cells 317
Glucose phosphorylation 318
The energy-rich intermediate ATP 191 Metabolic pathways for glucose 319
Oxidation-reduction 192 Metabolism of other hexoses 345
Biological oxidations 196 Biosynthesis of oligosaccharides in glycoproteins 350
Respiratory chain 197 Summary 354
Oxidative phosphorylation 204 Further reading 358
Phosphorylation at substrate level 215
Other electron transport systems 216 15. Lipid metabolism
Summary 217
Further reading 218 Plasma lipoprotein metabolism 360
Fat metabolism 367
10. Antioxidants Fatty acid biosynthesis 376
Eicosanoid biosynthesis 383
Reactive species 221 Triacylglycerol biosynthesis 386
Summary 230 Phospholipid biosynthesis 387
Further reading 230 Congenital disorders of complex lipid catabolism 389
 Contents ix
Cholesterol metabolism 390 20. Metabolism in some tissues
Cholesterol biosynthesis 390
Alterations of plasma lipids 395 Liver metabolism 483
Summary 397 Skeletal muscle metabolism 492
Further reading 400 Heart metabolism 496
Adipose tissue metabolism 496
16. Amino acid metabolism Nervous tissue metabolism 498
Summary 498
Essential AA 404 Further reading 500
Amino acid catabolism 408
Metabolic pathways of ammonia 411 21. The genetic information (I)
Other general mechanisms of amino acid metabolism 419
Metabolic pathways of AA 422 DNA replication 501
Summary 433 Transcription 511
Further reading 435 Molecular biology 517
Summary 531
17. Heme metabolism Further reading 533

Heme biosynthesis 437 22. The genetic information (II)

Catabolism of heme 441
Summary 446 Protein biosynthesis 535
Further reading 447 Mechanism of protein synthesis 544
Genetic mutations 552
18. Purine and pyrimidine metabolism Oncogenes 561
Epigenetics 563
Purine biosynthesis 449 Summary 565
Purine salvage pathway 451 Further reading 566
Purine catabolism 452
Uric acid 454 23. Regulation of gene expression
Gout 454
Pyrimidine biosynthesis 455 Gene regulation in eukaryotes 569
Pyrimidine catabolism 457 Gene regulation by noncoding RNAs 575
Di- and triphosphate nucleoside biosynthesis 458 Small noncoding RNAs 576
Deoxyribonucleotide biosynthesis 458 Long noncoding RNAs 579
Pharmacological approaches to purine and pyrimidine Riboswitches 579
metabolism 458 Summary 580
Summary 459 Further reading 581
Further reading 460
24. Posttranslational protein modifications
19. Integration and regulation of metabolism
Protein folding 583
Interconversion of carbohydrates, lipids, and amino Pathologies caused by misfolded
acids 461 proteins 584
Metabolic regulation 463 Posttranslational protein modifications 585
Summary 479 Summary 595
Further reading 481 Further reading 596
x Contents

25. Biochemical basis of endocrinology (I) Carbon dioxide transport 766

receptors and signal transduction Regulatory systems 768
Respiratory regulation 769
Receptors 598 Renal regulation 769
Signal transduction systems 610 Regulation of intracellular pH 772
Summary 625 Acid-base balance disorders 772
Further reading 627 H+ excretion regulation 774
Laboratory studies in acid-base disorders 774
26. Biochemical bases of endocrinology (II) Summary 775
hormones and other chemical intermediates Further reading 777

Hormones 629 29. Essential minerals

Endocrine glands and their hormones 632
Placental hormones 646 Macrominerals 779
Thyroid hormones 647 Trace elements 800
Adrenal gland hormones 652 Summary 808
Pancreatic hormones 664 Further reading 810
Gonadal hormones 676
Parathyroid gland hormones 684 30. Molecular basis of immunity
Kidney hormones 688
Heart hormones 690 Immune system 811
Gastrointestinal hormones 691 Innate system 811
Pineal gland hormone 693 Adaptive system 812
Eicosanoids 694 Genetic diversity of immunoglobulins 820
Growth factors 695 Complement 826
Nervous system chemical intermediaries Cellular immunity 831
(Neurotransmitters) 696 Cytokines 839
Summary 700 Vaccines 844
Further reading 705 Summary 845
Further reading 849
27. Vitamins
31. Hemostasis
Fat-soluble vitamins 710
Water-soluble vitamins 726 Blood coagulation 851
Summary 749 Coagulation disorders 858
Further reading 751 Summary 859
Further reading 860
28. Water and acid-base balance
32. Cell death
Water distribution in the body 754
Water balance 755 Apoptosis 861
Ionic composition of body fluids 756 Summary 868
Alterations of the water balance 762 Further reading 869
H+ concentration in body fluids 764
Regulation of H+ concentration 765 Index 871
 Preface

This is the second edition of the English resulting from the rapid progress in the disci-
version of “Quı́mica Biológica,” a textbook pline are presented. However, from the enor-
published in Spanish by El Ateneo, Buenos mous amount of information available, only
Aires, Argentina. the most relevant material needs to be selected
When first conceived, this book was primar- taking into consideration the needs of the future
ily intended to provide introductory informa- physician or junior researcher. At the same time,
tion in the area of biochemistry to premedical it is critical that the revision maintains the pre-
and medical students. Over the years, the book sentation and discussion of the material in a
developed and has become a source of informa- didactic manner to facilitate the learning
tion for advanced medical and graduate stu- process.
dents. In addition, the book should also be In this edition, we have updated and
useful to students in the allied health professions. expanded on different topics, reorganized con-
Following the experience that we have tents to achieve a better organization of the con-
acquired by teaching medical and graduate stu- cepts, and added more links to clinical scenarios
dents over the years, our approach to this book that result from biochemical alterations. Our
was to start describing the basic concepts in bio- goal has continued to focus on maintaining the
chemistry, developing each topic into more textbook concise and to deliver information in
complex concepts, and incorporating those the simplest manner possible. To achieve this,
new discoveries that are well supported and besides improving the text, we have used multi-
accepted by the scientific community. Also, we ple colors in all figures, added new figures, and
have tried to adopt an integrative approach, included additional tables that summarize the
making frequent links of biochemistry to cell main concepts in an easy manner. We believe
and molecular biology as well as physiology, that the book presents in a straightforward
thus emphasizing the relevance of the concepts manner a comprehensive view of the different
to the clinic. Ultimately, our goal is to present biochemical processes that occur in the human
a comprehensive view of the different biochem- body in health and disease.
ical processes that occur in the human body in A textbook cannot be successfully finalized
health and disease. without the collaboration of other talented indi-
The good acceptance of the first English edi- viduals. We acknowledge the dedication and
tion of the book gave us the impetus to continue hard work of Tonio Blanco, who has carefully
with a second edition. Updating a biochemistry designed and colored all the figures of this
textbook for medical students poses a great chal- book, keeping in mind the most didactic
lenge. This requires that the latest discoveries approach to deliver the material. We also thank

xi
xii Preface

Vickie Blanco and Adrian Blanco for their help- Cover

ful editorial comments on the final version of
the book. The cover represents one of the subunits of
We now invite the reader to dive into the the tetrameric enzyme lactate dehydrogenase
world of biochemistry through this book, hop- (LDH) (EC 1.1.1.27, L-lactate: NAD oxide reduc-
ing that it will provide a great learning experi- tase). The basic structure is common to the dif-
ence and will spark the curiosity and interest ferent isoforms of LDH, including the C
for scientific research, thus helping us expand subunit of the LDH C4 isozyme specific to the
the frontiers of knowledge in medical spermatozoa discovered by A. Blanco and W.
biochemistry. H. Zinkham (Science 139, 601–602, 1963).
 Introduction

Biological Chemistry, the science that seeks to The discovery of the structure of biological
explain life processes at the molecular level, molecules has advanced the understanding of
comprises two broad areas: one studies the com- their functions and mechanisms of action and
ponents of living organisms and is called Static has helped in deciphering their biological roles
or Descriptive Biochemistry; the other investigates in organisms.
the chemical transformations that occur in bio- Dynamic Biochemistry. Countless chemical
logical systems and is referred to as Dynamic reactions continuously take place in living
Biochemistry. Both these areas have witnessed beings in what is known as metabolism. The
an amazing development in the last 100 years. study of these reactions is contemplated by
Descriptive Biochemistry. Due to the vast com- Dynamic Biochemistry. The earliest goals in this
plexity of living organisms, studying their com- area of Biochemistry were directed to decipher-
position has been an extremely challenging ing the changes that occur to substances
undertaking that has demanded immense ingested with the diet and to identify the origin
research efforts. Even the simplest unicellular of waste products excreted by the body. Later,
organism contains a myriad of substances and studies were focused to understand the biosyn-
an impressive amount of chemical reactions. thesis of different endogenous components of
The study of the components of cells and tis- the body.
sues requires isolation, purification, structure The advances made in the area of metabolism
determination, and characterization of their have been, in part, a consequence of the progress
functional properties. Initially, biochemists of enzymology. The study of enzymes, catalysts of
directed their attention to simple compounds, biochemical reactions, has been crucial in the
which could be readily removed from animals interpretation of biological phenomena.
or plants, or compounds that could be easily Most chemical conversions in living beings
obtained by degradation of more complex take place gradually, through a series of reac-
substances. tions or metabolic pathways which, through
As in other disciplines, the advancement of sequential steps, convert the original compound
Biological Chemistry has closely followed tech- into one or more final product/s.
nological developments. The availability of From the first observations in the second half
new equipment and methods with increasing of the 19th century, researchers have worked
sensitivity and higher resolution capabilities relentlessly to unravel the astounding diversity
has allowed researchers to study molecules of and complexity of chemical pathways. This
the highest complex organization. Isolation, resulted in the assembly of “metabolic maps,”
purification, and analysis of macromolecules which illustrate the intricate pathways that com-
(proteins, nucleic acids, and polysaccharides), pounds follow as they are chemically modified.
which were once unimaginable, have now Although these maps look like a chaotic net-
become customary in biochemical laboratories. work, each reaction proceeds in a highly ordered

xiii
xiv Introduction

and coordinated manner to serve the needs of fermentation. This unity in biology has allowed
each cell and the organism as a whole. Studies using simpler organisms as an approach to
on the regulation and cross talk between meta- understand the function of multicellular organ-
bolic pathways have uncovered numerous isms of higher complexity.
highly refined regulatory mechanisms which, The body as an energy converting machine.
through modulation of enzyme activity, pre- According to their energy requirements, living
cisely coordinate the flow of compounds along beings can be divided into autotrophic and hetero-
specific metabolic pathways as required. trophic. Plants are autotrophic, which means that
The nervous and endocrine systems play an they are able to synthesize complex organic
essential role in coordinating and integrating compounds (carbohydrates, fats, nucleic acids,
the function of many pathways in different tis- and proteins) starting from simple inorganic
sues of multicellular organisms. These systems substances (water, carbon dioxide, nitrogen,
operate in response to a variety of stimuli by and phosphates). The energy for this synthesis
releasing chemical messengers. These messen- comes from the sun (photoautotrophs) or from
gers include neurotransmitters, hormones, cyto- electron donors provided by inorganic chemical
kines, and other factors that target specific sources, such as ammonium, sulfur, and ferrous
intracellular or cell surface receptors. Once acti- iron (chemoautotrophs).
vated, these receptors trigger a cascade of signal In contrast, multicellular animals are hetero-
transduction mechanisms that will finally trophic, depending on the intake of compounds
induce cell-specific effects. Signal transduction produced by other organisms. Normally, carbo-
mechanisms have been shown to exhibit a strik- hydrates, lipids, proteins, vitamins, and min-
ing degree of complexity, displaying a series of erals that animals obtain from the diet
mediator molecules that interact in different undergo chemical changes that serve two main
ways to activate or inhibit particular events in purposes: (1) the transfer of the energy con-
the cell. tained in these substances and (2) the supply
The unity of the biological world. Despite their of raw materials for the synthesis of the organ-
great diversity, living beings show remarkable ism’s own molecules. This process of synthesis,
unity with respect to the structures and basic like other cellular activities, requires energy.
processes that govern their function. For exam- The chemical energy obtained from compo-
ple, although macromolecules, such as proteins nents of the diet can be transformed to fuel the
and nucleic acids, differ from species to species, multiple activities occurring within cells. For
and even from individual to individual, they this reason, living organisms can be considered
show an overall basic structure that follows true energy converting machines. Chemical
the same general plan. All proteins are com- reactions called exergonic, which are those that
posed of long chains of the same 20 fundamental are accompanied by the release of energy, are
units (amino acids), and nucleic acids are consti- frequently coupled with others that require
tuted by long strands resulting from the assem- energy (endergonic). Many of these reactions
bly of four basic structures (nucleotides). result in the production of new molecules that
Likewise, the mechanisms underlying cell can trap and store the released energy until it
metabolism also show great similarity even is needed. Among the many molecules that play
across phylogenetically distant species. For this role, adenosine triphosphate (ATP) is the most
example, the reactions by which human skeletal common carrier of readily usable chemical
muscle fibers obtain energy for contraction energy.
mimic the transformations that yeast and other Reproduction capacity. A distinctive property
microorganisms perform during the process of of living beings is their ability to reproduce
 Introduction xv
and create, over generations, other organisms new ethical and social challenges arose, which
that are similar to their predecessors in external require careful consideration by scientists and
features, internal structure, and physiological the general public.
characteristics. This is possible due to the trans- Scope of Biological Chemistry. Advances in Bio-
mission of heritable traits from cell to cell and logical Chemistry have opened new horizons
from parents to offspring, which are stored in and prompted the development of other disci-
the genetic information contained in deoxyribo- plines, such as Cell Biology. The expansion of
nucleic acid (DNA) found within chromosomes. Biochemistry into areas such as Molecular Biol-
This genetic information is ultimately expressed ogy and Cell Biology has made the boundaries
through the synthesis of proteins with unique between these disciplines less defined. At pre-
characteristics for each species and individual. sent, any Biological Chemistry textbook needs
The “code” or “language” of the genetic infor- to expand its scope “invading” into areas of
mation in DNA is universal and practically the knowledge that were originally foreign to it.
same for all living beings. This is another exam- Reductionism-Complexity. Scientific research
ple of the remarkable unity of the biological has provided rational explanations for the prop-
world, which points to the common origin of life erties and functions of many biomolecules.
on Earth. Today, Biochemistry has a solid experimental
Changes (mutations) that spontaneously basis, which resulted from a reductionist con-
appeared in the “genetic message” through mil- ception of the study and interpretation of phe-
lions of years have created the diversity of the nomena. This strategy has proven highly
living world that we see today and have shaped successful in acquiring new knowledge. From
the particular characteristics that each species studies carried out in isolated molecules to
acquired during evolution. those performed in reconstituted in vitro sys-
The progress made in recent decades on tems, scientists have been able to develop
understanding the structure and function of models in an attempt to describe different
DNA has been remarkable. This has allowed a in vivo processes.
better understanding of the mechanisms respon- As these models developed, it became appar-
sible for the transmission of genetic characteris- ent that biological systems are highly complex.
tics and the eventual modifications seen Paradoxically, achievements of reductionism
between an individual and its progeny. In addi- have shown its limitations. It is now clear that
tion, the understanding of DNA has opened living organisms cannot just be defined as the
unforeseen opportunities for applications in dif- sum of their components. The integrated oper-
ferent fields. Molecular Biology, which emerged ation of these components generates a func-
within Biochemistry, has made astonishing con- tional intricacy that makes the analysis and
tributions to our knowledge of the complete understanding of living beings extremely chal-
sequence of the DNA in the genome of humans, lenging. This is the stimulus that fuels scientific
plants, and animals. Also, this discipline has curiosity, drives the continuous search for
allowed the manipulation of genes; a technology learning biological phenomena, and contributes
that has direct application in biology and medi- to the constant expansion of the frontiers of
cine, including the production of genetically knowledge.
modified organisms with specific desired prop- Importance of Biological Chemistry. Without a
erties and the development of genetic therapeu- doubt, the progress of Biochemistry has been
tic approaches to treat a variety of diseases. one of the main factors that have contributed to
Along with the evident benefits brought the development of the biological sciences.
about by the advancement of molecular biology, Medical disciplines have benefited tremendously
xvi Introduction

from the advances in Biochemistry and it can be Chemistry, not only will they find the grounds
anticipated that this progress will continue at an for a rational interpretation of many physiolog-
even faster pace in the future. ical and pathological phenomena but also the
Biologists and physicians need to acquire a stimulus for a permanent search for new
solid background in basic sciences. In Biological knowledge.
 C H A P T E R

1
Chemical composition of living beings

Biogenic elements constitutes approximately 21% of the total

Earth’s weight.
Life emerged on Earth many millions of years Carbon belongs to the same group in the peri-
after the planet was first formed. Only a few ele- odic table as silicon and shares many of its prop-
ments within the inorganic matter of the Earth’s erties. However, carbon can form more stable
crust and atmosphere were selected as the build- chemical bonds long branched chains, double
ing blocks of all living organisms. These basic and triple bonds, covalent bonds with different
elements of life are called biogenic elements. atoms, and can adopt a variety of different spa-
Mammals, animals of great complexity, are tial conformations. This gives carbon the unique
composed of merely 20 elements, four of which potential to generate a variety of chemical com-
(oxygen, carbon, hydrogen, and nitrogen) are binations that are essential for the makeup of the
the most abundant ones, comprising approxi- molecules of living organisms.
mately 96% of the total body mass (Table 1.1). The selection of the other elements that
All elements existing in the human body, accompany carbon as components of the living
except for iodine (which has an atomic number matter depends on the size of these atoms and
of 53), are placed within the first four periods of their ability to share electrons in covalent bonds.
the periodic table and possess atomic numbers The smaller atomic size of these elements allows
lower than 34. Among the four most abundant, them to establish stable bonds and stronger
oxygen has the highest atomic number (8). molecular interactions.
While oxygen is relatively common on Earth, Taking into consideration their relative
the other biogenic elements are less abundant, amounts, biogenic elements can be classified
suggesting that they have properties that gave into three main categories:
them a selective advantage in forming the basic
units of life. For example, carbon and not silicon 1. Primary elements. These include oxygen,
has been the element around which life devel- carbon, hydrogen, nitrogen, and the less
oped, even though silicon is widespread and abundant, calcium and phosphorus.

Medical Biochemistry 1 Copyright # 2022 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/B978-0-323-91599-1.00025-0
2 1. Chemical composition of living beings

TABLE 1.1 Elements of the human body and their biological importance, including hemoglobin.
relative abundance. Sulfur is present in almost all proteins and
other molecules of biological interest.
Primary elements
3. Trace Elements. These are also known as micro
Oxygen 65.0 Nitrogen 3.0 constituents or oligoelements, because they
Carbon 18.5 Calcium 1.5 are present in the body in very small
quantities.
Hydrogen 10.0 Phosphorus 1.0
Secondary elements Iodine is a constituent of the thyroid hor-
mone. The other oligoelements (Cu, Mn, Co,
Potassium 0.30 Chlorine 0.15
Zn, Mo, and Se), while also scarce, are vital for
Sulfur 0.25 Magnesium 0.05 the normal function of the body and most are
Sodium 0.20 Iron 0.005 essential factors for the proper activity of
enzymes.
Oligoelements
Fluorine 0.001 Zinc Traces
Biological compounds
Cuprum 0.0002 Cobalt Traces
Iodine 0.00004 Molybdenum Traces The elements mentioned earlier are found as
part of different inorganic or organic com-
Manganese 0.00003 Selenium Traces
pounds. Among the inorganic compounds, water
Values are expressed as a percent of total body mass. is of exceptional importance, not only because it
exists in large quantity (comprising 65% of the
Together, these six elements account for more total body weight of an adult individual), but
than 98% of the total body mass. Oxygen and also due to its numerous biological roles. Also,
hydrogen form water, the most abundant of great significance are minerals, such as cal-
substance in the body. Carbon, oxygen, cium phosphate, which constitute the nonsolu-
hydrogen, nitrogen, and phosphorus form ble components of bones and teeth. The
part of a variety of essential organic remaining inorganic components dissolved in
molecules. Calcium, in combination with body fluids and in the cytoplasm of cells play
other substances, is mainly found in bone, critical roles in many body functions as ions.
and in its ionic state, is involved in numerous Within the organic compounds, Carbon is a
physiological processes. key component in most of the solid substances
2. Secondary elements. The elements in this group of the body, forming part of molecules of high
include potassium, sulfur, sodium, chlorine, biological relevance, such as proteins, carbohy-
magnesium, and iron. They are found in drates, lipids, and nucleic acids. There are other
much lower relative quantities than those of compounds, such as vitamins, hormones, and
the previous group and they exist as salts, pigments, that also have critical roles.
inorganic ions, and form part of different Table 1.2 provides a general view of the relative
organic molecules. chemical composition and approximate
Na+ and Cl are the main extracellular amounts of inorganic and organic compounds
ions, while K+ is the main intracellular ion. in some human tissues.
Mg2 + is necessary for many reactions carried The chapters that follow will describe in more
out by biological catalysts or enzymes. Iron is detail the structure and properties of the main
an essential component of substances of high substances that compose the human body.
 Further reading 3

TABLE 1.2 Chemical composition of human tissues. primary elements, they exist as free ions or
form salts and form part of different body
Muscle Bone Brain Liver molecules. Na+ and Cl are the main ions in
Water 75.0 22.0 77.0 70.0 the extracellular space, while K+ is the main
intracellular ion. Mg2+ is indispensable for
Carbohydrates 1.0 Scarce 0.1 5.0
many reactions catalyzed by enzymes. Iron is
Lipids 3.0 Scarce 12.0 9.0 an essential component of substances, such as
Proteins 18.0 30.0 8.0 15.0 hemoglobin. Sulfur is present in almost all
proteins.
Other organic 1.0 Scarce 1.5 1.0
substances 3. Trace elements, microconstituents, or
oligoelements, are present in the body in
Other inorganic 1.0 45.0 1.0 Scarce
scarce quantities. Iodine is present in the
substances
thyroid hormone. Cu, Mn, Co, Zn, Mo, and Se
Values are expressed as a percent of total tissue mass. are essential for the normal function of
enzymes.
Summary Biological compounds include inorganic and
organic substances. Among inorganic
Carbon has been the element around which compounds is water, the solvent in body fluids
life developed. This is due to the capacity of car- and tissues. It comprises 65% of the total body
bon to form stable chemical bonds, long weight of an adult individual. Nonsoluble
branched chains, double and triple bonds, cova- inorganic compounds have different roles, for
lent bonds with different atoms, and a variety of example, calcium phosphate is an essential com-
different spatial conformations. ponent of bone. Organic biological compounds
Biogenic elements include a series of sub- include proteins, carbohydrates, lipids, and
stances, which can be divided into the following nucleic acids. Others, such as vitamins, hor-
groups: mones, and pigments perform critical roles,
1. Primary elements include oxygen, carbon, and all contain carbon as the key component.
hydrogen, nitrogen, and the less abundant,
calcium and phosphorus. They comprise Further reading
98% of the total body mass and constitute all
de Duve, C., 1995. The beginnings of life on Earth. Am. Sci.
the essential body molecules and water.
83, 428–437.
2. Secondary elements comprise potassium, Morowitz, H.J., 2002. The Emergence of Everything (How the
sulfur, sodium, chlorine, magnesium, and World Became Complex). Oxford University Press,
iron. While in much lower amounts than Oxford.
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 C H A P T E R

2
Water

Water is a tasteless, colorless, odorless, and applied in therapeutic or cosmetic surgeries to

transparent inorganic substance. It is abundant eliminate abnormal cells (cryosurgery). On the
on Earth, covering approximately two-thirds other hand, freezing with cryoprotectants,
of its surface, where it is most commonly found which avoid crystallization of the cell plasma
in a liquid state. It is the primordial fluid of membrane, allows for the maintenance of cells
living organisms and the solvent in which all and biological materials during extended
biological solutes are dissolved. There is no periods. This approach is commonly used in
biological process that occurs independently many laboratories for the long-term storage of
from the direct or indirect participation of water. cell lines, sperm, oocytes, and embryos.
Water is the most abundant component of the Water has exceptional properties. For example,
human body; it constitutes approximately 65% at a pressure of 1 atm, its freezing (0°C or 32°F)
of the weight of an adult individual. and boiling temperatures (100°C or 212°F), and
Water undergoes phase transitions depend- its heat of vaporization (40.71 kJ/mol) are signifi-
ing on temperature changes. These include cantly higher than those of other compounds of
freezing (water to ice), melting (ice to water), similar molecular mass. These, and other features
vaporization (water to vapor), condensation of water, depend on the particular molecular
(vapor to water), sublimation (ice to vapor), structure of this compound.
and deposition (vapor to ice). In the water molecule (H2O), oxygen (O) is
In the human body, at 37°C, water is pre- bound by simple covalent bonds to two hydro-
sent in its liquid form, comprising approxi- gen (H) atoms. As O is more electronegative
mately 65% of the body weight of an adult than H, the pair of electrons shared in each
individual. of the bonds is closer to the nucleus of the
If the temperature is decreased, water crystal- O atom. This creates a partial electronegative
lization can severely damage the cells. This is charge in the vicinity of the O nucleus and
due to the direct mechanical effect of the water an electropositive charge around each H,
crystals as well as the osmotic effect that they which remains almost reduced to a “naked”
exert, both of which disrupt the cell membranes. proton. Individually, the OdH bonds are
The use of subzero temperatures has been polar (covalent polar).

Medical Biochemistry 5 Copyright # 2022 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/B978-0-323-91599-1.00027-4
6 2. Water

The water molecule is polar

If the three atoms of a water molecule were
distributed linearly (HdOdH), the resulting
“center of gravity” of the positive charges would
be located in the center of the molecule, coincid-
ing with the position of the negative charge. In
this case, the molecule would be nonpolar, as
occurs with other compounds that have polar
covalent bonds, such as carbon dioxide (CO2) FIG. 2.4 Hydrogen bond between two water molecules.
or carbon tetrachloride (CCl4). However, in
water, the two OdH bonds are not in a straight
line but form an angle of 104.5 degrees (Figs. 2.1 Hydrogen bond
and 2.2). The negative charge is located around
the vertex of the molecule, while the resultant of The hydrogen bond is not exclusive to water
the positive charges is conceivably located at the molecules; as it will be discussed in the follow-
midpoint of the line joining the two hydrogen ing chapters, it is also found in other compounds
nuclei. This creates a dipole, allowing the mole- of great biological importance.
cule to behave as polar, although it is electrically The hydrogen bond is easily established
neutral (Fig. 2.3). between an electronegative atom (typically
The polarity of water molecules enables them to O or N) and an H atom covalently bonded to
attract each other electrostatically. The partial pos- another electronegative atom. The binding is
itive charge of one H in a molecule is pulled toward most stable when the three components
the negatively charged O of another molecule, involved, the two electronegative atoms and
thereby establishing a hydrogen bond (Fig. 2.4). the intermediate H, are on the same line (Fig. 2.4).
The hydrogen bond allows the interaction
among water molecules and explains the unique
properties of this substance. The behavior of
water does not correspond to that expected from
FIG. 2.1 Position of atoms in the water molecule. a compound with the chemical formula H2O,
but to a polymeric complex (H2O)n, which water
adopts when it is in its solid and liquid states.
A water molecule can theoretically be con-
ceived as a tetrahedron. In this configuration,
the oxygen atom is in the center, the OdH bonds
are directed toward two of the vertices of the tet-
FIG. 2.2 Water molecule. Left: model of spheres and rods; rahedron, and the nonshared oxygen electrons
right: a space filling spatial model, with oxygen in red.
are located in sp3 hybrid orbitals, which are ori-
ented toward the other two vertices of the tetra-
hedron (Fig. 2.5). This arrangement allows each
water molecule to form hydrogen bonds with
four other molecules of water (Fig. 2.6).
In a regular tetrahedron, the angle between
FIG. 2.3 Distribution of electric charges in the water shared pairs of electrons between O and
molecule. H should be 109.5 degrees. In the water
 Water as solvent 7
water. At increasing pressure, various crystal-
line forms of water can be produced, which
are characterized by different shapes and densi-
ties. Under particular temperature and pressure
conditions, solid water can also form an amor-
phous structure. This does not form spontane-
FIG. 2.5 Tetrahedral geometry of the water molecule. ously on Earth, since it requires very fast
freezing or the application of high pressure, so
the water molecules do not have time to form
crystals. In addition, different types of amor-
phous ice can be found depending on its density
(low, high, and very high-density amorphous
ice). Amorphous ice is used in laboratories for
cryogenic electron microscopy (cryo-EM),
because it allows outstanding preservation of
biological samples for imaging, keeping cells
in a state close to that in liquid water.
In liquid water, the crystal lattice order is lost.
Although water molecules are associated with
each other (on average, it is calculated that each
water molecule is connected to the other 3.4
water molecules), the hydrogen bonds that hold
them together are very unstable, constantly
forming and breaking apart. The molecular
FIG. 2.6 Association of water molecules. Hydrogen
water clusters are therefore “fluctuating” or
bonds (red dotted line) result from the tetrahedral arrange-
ment of the water molecule. “oscillating.” This dynamic interaction between
water molecules accounts for the high heat of
vaporization and high boiling point of water.
molecule, the two pairs of electrons involved in These intermolecular attractions, which do not
covalent bonding with H are pushed away by exist in other substances of similar molecular
the nonbonding electron pairs. These nonbond- mass, are strong and a large amount of energy
ing electron pairs are closer to the oxygen atom is required to break them. The hydrogen bonds
and cause a slight distortion of the tetrahedron, among water molecules also explain the higher
resulting in an angle of 104.5 degrees between surface tension and viscosity of water compared
the OdH bonds. to that of most other organic liquids. However,
In its solid form (ice), water adopts the tetra- due to the continuous fluctuation of the hydro-
hedral configuration allowing it to produce a gen bonds, liquid water has more fluidity and
regular crystal lattice. The molecules remain at lower viscosity, than other polymeric molecules.
fixed distances from each other, which are deter-
mined by the length of the hydrogen bonds. The
ice crystal lattice has relatively more void space Water as solvent
than liquid water. In liquid water, the molecules
not associated with H bonds have more freedom The polar property of water is responsible for
and can become closer to each other at 0.45 nm. the many interactions that it establishes with
This explains why ice is less dense than liquid other substances. The type of interaction that
8 2. Water

water establishes varies and depends on the As ionic and nonionic polar compounds
nature of the other substance. interact with water, they are hydrophilic and
Ionic compounds are, in general, soluble in capable of forming stable water solutions.
water. For example, NaCl crystals have electro- Nonpolar compounds, such as hydrocarbons,
static attractions between the Na+ and Cl
ions are practically insoluble in water. This is due
that maintain the highly ordered lattice of this to the lack of attraction between these molecules
salt. When NaCl crystals come in contact with and water. They are hydrophobic and, in gen-
water, the organization of the molecules of both eral, they dissolve well in nonpolar or slightly
compounds is altered. The attraction between polar organic solvents (benzene, carbon tetra-
the dipolar water molecules and the Na+ and chloride, and chloroform). Nonpolar molecules
Cl
ions has enough strength to dissociate the can establish mutual attractions through what
ions from the salt, eventually separating and dis- is known as hydrophobic interactions.
persing them into the solvent. The ions in an Amphipathic or amphiphilic compounds, such as
aqueous solution become hydrated or sur- phospholipids or monovalent metal salts of
rounded by water molecules, forming a layer long-chain fatty acids (sodium or potassium
or “halo” around them. Inorganic cations (Na+, soaps), possess hydrophobic and hydrophilic
K+, Ca2+, and Mg2+) and organic amine groups groups within the same molecule (Fig. 2.9A).
(C-NH3+) attract the negative charge of water Upon contact with water, they orient their
molecules. Inorganic anions (Cl
, HPO4 2
, hydrophilic moiety toward the water phase
HCO3
) and organic carboxylate (dCOO
) and their nonpolar portion away from the aque-
attract the positive charge of the dipole (Fig. 2.7). ous phase (Fig. 2.9B). When these molecules are
Nonionic polar compounds, such as alcohols, in water, they can form spherical clusters called
aldehydes, or ketones, form hydrogen bonds micelles. Nonpolar chains of the amphipathic
with water via their hydroxyl or carbonyl compounds span toward the interior of the
groups (Fig. 2.8). This facilitates the solubility micelle, mutually attracted by hydrophobic
of nonionic polar compounds in water. interactions. In contrast, the hydrophilic ends
of amphipathic molecules interact with the
aqueous phase (Fig. 2.9C), preserving the stabil-
ity of the micelles.

Water as an electrolyte
FIG. 2.7 Interaction of ions with water molecules. Substances that dissociate into charged
Hydrated Na+ is shown on the left and hydrated Cl
on particles or ions in an aqueous solution are
the right.
called electrolytes, and the solutions they
form allow the passage of an electric current
through them.
Solutions containing equal concentrations of
different electrolytes may have different capaci-
ties to conduct electrical current. Some are excel-
lent conductors, while others are poor.
According to this criterion, electrolytes can be
divided into strong and weak.
FIG. 2.8 Hydrogen bond (red dotted line) between water For example, a 1 M solution of HCl is a much
molecules and an alcohol (top), or a ketone (bottom). better conductor than a 1 M solution of acetic
 Equilibrium constant 9

FIG. 2.9 (A) Scheme of amphiphilic or amphipathic molecules. (B) Arrangement of amphiphilic molecules in the water-air
interface. (C) Amphiphilic molecules forming a micelle in water.

acid (CH3COOH). The capacity to carry an AB, this reactant will partially ionize into the
electrical current depends on the availability of products, A and B ions, as follows:
ions in the solution. As both the HCl and acetic
acid 1 M solutions have the same number of mol-
ecules per liter (1 mol of substance or 6.022  1023 The dissociation of AB into ions continues until
molecules), the higher conductivity of the HCl equilibrium is reached, in which whole mole-
solution must be due to a greater number of ions cules and ions coexist in the solution at relatively
in this solution than in the acetic acid solution. constant amounts.
This shows that HCl ionizes more than acetic The concentration of ions and of entire mole-
acid. HCl is a strong electrolyte, while acetic acid cules at equilibrium depends on the nature of
is a weak electrolyte, in which only a small por- the electrolyte, the initial concentration of the
tion of the total molecules dissolved in water substance, and the temperature. For a system
separate into ions. in chemical equilibrium at a given temperature,
there is a constant numerical relationship
between its components which is always satis-
Equilibrium constant fied regardless of the initial concentration of
the substance. This relationship is the equilibrium
The ionization process for dissolved electro- constant (Keq) represented by the following
lytes can be compared to a chemical reaction. equation:
For strong electrolytes, it can be considered that
½ A +  ½ B

almost all molecules dissociate into ions and the Keq ¼
½AB
reaction proceeds in one direction:
[A+], [B
], and [AB] represent concentrations of
HCl ! H+ + Cl

A+, B
, and AB, respectively.
For weak electrolytes, the reaction is reversible. The equilibrium of a weak electrolyte is
If one considers the weak electrolyte molecule dynamic, with molecules constantly ionizing
10 2. Water

and ions merging together to form the original Equilibrium of water ionization
molecules. In equilibrium, both processes occur
at the same rate; therefore, the concentration of Water ionizes very weakly generating hydro-
each component in the system remains gen ions or protons and hydroxyl ions according
unchanged. to the reaction:
In the ionization reaction of the weak electro-
lyte AB:
The representation of H+ as a free ion in water is
not strictly correct, since protons rapidly react
The velocity (V1) of the ionization reaction (reac- with nonionized water molecules. To depict a
tion 1) will be: more accurate view of the state of H+, many
authors refer to it as hydronium ion (H3O+).
V1 ¼ k1 ½AB Even more precise is the notation [H(H2O)n]+.
where k1 is a constant value that is unique to However, for practical purposes, we will con-
each substance at a given temperature and tinue using H+ to represent the H ions but recog-
[AB] is the molal concentration of AB. nizing that they do not exist independently.
For the reverse reaction (2), the velocity V2 at The water ionization constant is expressed by
which A+ and B
associate to form AB is: the following equation:
V2 ¼ k2 ½A+  ½B
 ½H+  ½OH

Keq ¼
½H2 O 
In equilibrium, the rates of the forward and
The Keq value of pure water has been deter-
reverse reactions are equal (V1 ¼ V2), therefore:
mined by measuring its electrical conductivity.
k1 ½AB ¼ k2 ½A+  ½B
 By knowing the conductivity of the ions, it is
possible to calculate the concentration of the free
then:
ions present in water. In pure water at 25°C, the
k1 ½A+  ½B
 [H+] is 0.0000001 or 10
7 M and the [OH
] is the
¼ same since water ionization generates equal
k2 ½AB
quantities of OH
and H+.
k1/k2 is a ratio of two constants, which results in The Keq value for pure water is very low and
another constant, designated K or Keq, therefore: varies with temperature. For example, at 25°C,
½A+  ½B
 water Keq is 1.8  10
16 M, while at 37°C it is
Keq ¼ 4.3  10
16 M. According to these figures, water
½AB
should not be considered an electrolyte. Why
This constant gives an idea of the degree of ion- so much attention is placed on the H+ generated
ization of an electrolyte. Weak electrolytes have by water ionization when its Keq is so small?
a low Keq value. A Keq close to one indicates that Changes in [H+] often have important biological
the electrolyte is highly ionized. When Keq is effects. Hydrogen ions are very small (they con-
approximately 10, the electrolyte is practically sist of a proton) and have, in comparison to other
completely ionized. In biological systems, an ions, a large charge density, which creates a sig-
electrolyte can be considered strong when Keq nificant electrical gradient around them. This
is greater than 0.0001 or 10
4. A substance with affects the hydrogen bonds that help maintain
a Keq lower than 10
14 is not considered an elec- the structure and conformation of biologically
trolyte. Weak electrolytes have Keq that vary important macromolecules. The ionization state
between 10
4 and 10
14. of functional groups, critical for the function of
 Acids and bases 11
certain compounds dissolved in water, is also value of the ionic product will tend to be
affected. restored by increasing the rate of H+ and OH

Since the molecular mass of water is 18 Da association to form whole water molecules. Con-
(1 mol of water ¼ 18 g), the molal concentration sequently, the concentration of OH
ions
of water molecules in pure water is approxi- decreases until equilibrium is reached when
mately 55.55 (in 1000 g of water there are the value of Kw is 10
14 (at 25°C). Thus, if the
1000/18 ¼ 55.55 mol). Only 0.0000001 mol of the HCl added increases the H+ concentration to
total 55.55 mol are ionized, which means that 10
1 M (1 million times higher than that of pure
only 1 molecule every 555,500,000 is ionized. water at 25°C), the OH
must be reduced to a
Then, the water ionization constant can be repre- value of 10
13 (1 million times lower than that
sented by the following equation: of pure water). Therefore, the ion product will
remain constant:
K ½H2 O ¼ ½H+  ½OH

As the amount of ionized molecules is negligible Kw ¼ 10
1  10
13 ¼ 10
14
compared to the total number of molecules, it An analogous adjustment in the concentration of
can be considered, without making any appre- H+ ions occurs if the electrolyte that is dissolved
ciable error, that the ionization process does in water increases the concentration of OH

not modify the concentration of nonionized mol- (NaOH ! Na+ + OH
). The increase in [OH
]
ecules [H2O]. Therefore, the first term of the will produce a decrease in the concentration of
equation is the product of two constants (K H+ by forming whole water molecules (H2O)
and [H2O]), which is a new constant, designated to maintain the Kw value.
Kw:
K½H2 O ¼ Kw ; Kw ¼ ½H+  ½OH

Acids and bases
The Kw constant is designated ion product for
water; its value at 25°C is: A solution is neutral when the hydrogen ion
concentration is equal to the hydroxide ion con-
Kw ¼ ½H+  ½OH
 ¼ 0:0000001  0:0000001
centration. Pure water is neutral because [H+] ¼
¼ 10
7  10
7 ¼ 10
14
[OH
]. At 25°C, pure water has 1  10
7 M [H+]
In pure water, [H+] ¼ [OH
]. So, [OH
] can be and 1  10
7 M [OH
], but this markedly changes
replaced by [H+] in the equation, or vice versa: with temperature. For example, at 0°C [H+] or
pffiffiffiffiffiffiffi [OH
] ¼ 3.4  10
8 and at 100°C, 8.8  10
7.
Kw ¼ ½H+  ½H+  ; ½H+  ¼ Kw pffiffiffiffiffiffiffi
When the concentration of hydrogen ions in a
or Kw ¼ ½OH
 ½OH
 ; ½OH
 ¼ Kw
solution is greater than that of hydroxide ions, it
Kw is a constant value in pure water and in any is considered acidic. On the other hand, when the
aqueous solution. Any increase in the concentra- hydrogen ion concentration is lower than that of
tion of one of the two ions of water will cause an hydroxide ions it is deemed basic or alkaline.
immediate decrease in the concentration of the These concepts lead to some practical defini-
other ion, shifting the equilibrium toward the tions regarding acids and bases. Acids are sub-
formation of whole water molecules; the ion stances that increase [H+] when dissolved in
product remains unchanged. If an electrolyte water or aqueous solutions. Conversely, bases
ionizes and provides hydrogen ions when dis- are substances that decrease [H+] when dis-
solved in water, as seen with HCl solved in water or aqueous solutions.
(HCl ! H+ + Cl
), the H+ concentration will According to the Brønsted and Lowry acid–
increase compared to that of pure water. The base theory, acids are compounds or ions with
12 2. Water

the ability to release protons (H+) into the concentration for the same acid concentration
medium, while bases are those which accept will be greater in the solution of a strong than
protons from the medium. a weak acid.
Here, the definition of acids is extended to The ability of an acid to lose protons (acid
ions, such as HSO


4 or NH4 , which can transfer strength) is expressed by its ionization constant
+
one H ion to the solution. Usually, the hydrox- (Ka). If we consider an acid HA:
ides of alkaline metals are considered typical
½H+  ½A

bases (i.e., NaOH), and strictly, according to Ka ¼
the Brønsted-Lowry acid-base theory, the base ½HA
is the OH
that those compounds release into The larger the Ka, the easier the acid releases pro-
the solution. When an OH
accepts H+ from tons. In general, the Ka of strong acids reaches
the solution, it will result in the formation of values that are very large (102 to 1010) and for
water molecules. All ions that can capture H+, practical purposes, it is not taken into account.
such as Cl
or CO2
3 , are considered bases. Strong acids are considered to be completely
When a molecule or anion accepts H+ (also ionized in dilute aqueous solutions. Weak acids
known as a Brønsted-Lowry base), a are partially ionized and their Ka constants are
“conjugated acid” of that molecule is formed. very small.
Bases can also be divided into strong [NaOH,
Conjugated
KOH, Ca(OH)2, etc.] and weak (NH3, trimethy-
Base Proton acid
lamine, aniline, etc.). Strong bases ionize
OH
+ H+ ! H2O completely in a solution. Similar to weak acids,
NH3 + H+ ! NH+4 the ionization constants of weak bases (Kb)
CO2

3 + H+ ! HCO
3 reflect their degree of ionization.
A useful generalization regarding the relative
strength of the acid-base pair is: if acid is strong,
When an acid loses H+, its “conjugated base” is its conjugate base is weak and if a substance is a
formed. strong base, its conjugate acid is weak.
Conjugated
Acid Proton acid pH
HCl + H +
! Cl
H2SO4 + H+ ! HSO
4
As the ion product of water [H+]  [OH
] has
HNO3 + H+ ! NO
3
a constant magnitude at a given temperature, it
is sufficient to know the concentration of one of
the ions to deduce the concentration of the other.
Usually, the concentration of hydrogen ions is
The strength of acids and bases used. However, the small amount of free [H+]
makes its use cumbersome; simpler ways to
The strength of an acid or a base is deter- express [H+] have been proposed. In 1909 the
mined by its tendency to lose or gain protons. Danish biochemist P.L. Sørensen proposed pH
Just like electrolytes, acids can also be divided as the notation for the [H+] of a solution. The
into strong (HCl, H2SO4, HNO3) and weak term pH received wide acceptance and is today
(H2PO
4 , CH3dCOOH, H2CO3) acids. When the most commonly used expression to indicate
dissolved in water, strong acids ionize almost the concentration of hydrogen ions.
completely, while weak acids only ionize in a To obtain the pH value of a solution, a double
small proportion. Consequently, the hydrogen transformation is required. This includes taking
 Buffers 13
the reciprocal of the [H+] and then, the logarithm of 7.5 at 0°C and 6.1 at 100°C. At 37°C, the
of that value. In this manner, pH can be defined human body temperature, neutral pH is 6.8.
as the logarithm of the reciprocal of the hydro- 3. Usually, the pH of a solution varies between 0
gen ion concentration or, in other words, the and 14. These limits of pH cover the range of
negative logarithm of the H+ concentration. [H+] from a 1 M strong acid solution (pH ¼ 0) to
a 1 M strong base solution (pH ¼ 14). However,
1
pH ¼ log ¼
log ½H+  in theory, the pH scale can extend beyond
½H+  these limits. The lowest value of [H+] that can
In the case of pure water at 25°C [H+] ¼ [OH
] be obtained in aqueous solutions is 10
15 M
¼10
7 M, therefore: and the highest is 15 M, corresponding to pHs
of 15 and
1.2, respectively.
1 1
pH ¼ log ¼ log
7 ¼ log 107 ¼ 7
½H+  10
The same notation can be used for other param- Buffers
eters, including [OH
], Kw, and Ka, which will
render pOH, pKw, and pKa, respectively. Buffers are systems that reduce the changes in
hydrogen ion concentration in a solution when
1 1 acid or alkaline electrolytes are added. In other
pOH ¼ log pKa ¼ log
½OH
 Ka words, buffers can minimize the pH deviations
There are several points related to the pH nota- produced by acids or bases in a given medium.
tion to be taken into account: In general, a buffer solution consists of a mix-
ture of a weak electrolyte (acid or base) and the
1. No direct relationship exists between the corresponding salt of that acid or base, which
magnitudes of [H+] and pH. When the value acts as a strong electrolyte. Examples of buffer
of [H+] increases, the pH decreases, and vice systems include:
versa. Moreover, since the pH scale is
logarithmic (not arithmetic, such as [H+]), any Carbonic acid
sodium bicarbonate
change in pH indicates a 10-fold change in ðH2 CO3 =NaHCO3 Þ
[H+]. For example, a change in 2 pH units Acetic acid
sodium acetate
indicates a 100-fold change in [H+], a pH ðCH3 COOH=CH3 COONaÞ
change of 3 shows a 1000-fold change in [H+], Monosodium phosphate
disodium
and so on (Fig. 2.10). phosphate ðNaH2 PO4 =Na2 HPO4 Þ
2. A pH of 7 indicates neutrality only at 25°C. As Ammonia
ammonium chloride
the [H+] changes with temperature, so does ðNH3 =NH4 ClÞ
the value of pH. Pure water (neutral) has a pH

FIG. 2.10 Correlation between [H+] and pH values.

14 2. Water

Mechanism of buffers action. Carbonic acid in solu- the solution is very low and the pH remains
tion ionizes into bicarbonate (HCO
3 ) and hydro- almost unchanged.
gen (H+) ions according to the following If a base (e.g., NaOH) is added to a buffered
reaction: system, the OH
ions bind to the hydrogen ions
in the solution to produce water. This causes a
The acid ionization constant (Ka) is given by the shift in the equation to the right, favoring car-
relationship: bonic acid ionization, generating new hydrogen
  + ions that combine with OH
:
HCO
3 ½H 
Ka ¼
½H2 CO3  NaOH + H2 CO3 ! HCO
+
3 + Na + H2 O
Being a weak electrolyte, carbonic acid has a low Consequently, the increase of OH
concentra-
capacity to ionize in water; it produces a very tion in the solution is small and the pH is altered
small number of ions compared to the total num- only slightly.
ber of whole carbonic acid molecules. When a pH of buffer solutions. In a system consisting of
salt of the same acid is added to this solution, a weak acid and its salt, the pH can be calculated
a buffer system is formed. Sodium salt is a from the ionization constant of the acid and the
strong electrolyte that completely ionizes into initial concentration of the salt.
bicarbonate and sodium: Let’s consider a buffer consisting of a weak
acid (HA) and its salt (NaA). The weak acid dis-
NaHCO3 ! Na+ + HCO

3 sociates according to the equation:

Both components of the system release bicar- HA ! A
+ H+

bonate ion when they ionize. As HCO
3 is added The Ka of the system will be:
to this system, the Ka value of the acid must be
maintained. A shift in carbonic acid toward ½A
 ½H+ 
Ka ¼
the formation of nonionized NaHCO3 molecules ½HA
takes place, decreasing the ionization of H2CO3
The salt acts as a strong electrolyte and dissoci-
to the point that it can be considered practically
ates according to the equation:
in all the nonionized states. A new equilibrium
is achieved that is characterized by a high con- NaA ! A
+ Na+
centration of bicarbonate ions and acid mole-
The salt is completely dissociated from the solu-
cules, and by a low concentration of hydrogen
tion and the concentration of anions (A
) and
ions. When a strong acid (HCl) is added to this
cations (Na+) is equal to the initial concentration
solution, the increase in hydrogen ions shifts
of the salt. As the anion A
is common to both
the equilibrium to the formation of whole car-
acid and salt, its high concentration in the solu-
bonic acid molecules, and its concentration
tion (from total ionization of the salt) causes a
increases. Concomitantly, the amount of bicar-
displacement of the acid ionization toward the
bonate ions decreases.
formation of nondissociated molecules. Virtu-


HCl + HCO

3 ! H2 CO3 + Cl
ally all of the acid in the solution is nonionized.
Hence, the concentration of acid molecules can
In other words, a large portion of the hydrogen be considered equal to the initial concentration
ions from the hydrochloric acid is taken up by of the acid.
the bicarbonate ion generating nonionized car- In the acid ionization equilibrium equation,
bonic acid. Therefore, the increase in [H+] in [A
] can be replaced by the initial concentration
 Titration curve of acids and bases 15
of salt and the concentration of HA by the initial number of equivalents of acid and base combine,
concentration of the acid: a point of equivalence is reached.
As the reaction proceeds, once the point of
½salt½H+ 
Ka ¼ equivalence is surpassed, the medium shows
½acid progressive changes in pH. Any additional
Then, the [H+] is: small amount of acid or base added to the solu-
tion causes a marked pH change. The pH values
½acid at each step of the titration are determined by
½H+  ¼  Ka
½salt potentiometric methods.
The logarithm of the reciprocal of [H+] is: The titration process can be followed by using
what is known as neutralization or titration
1 ½salt 1 curves. These represent the relationship
log + ¼ log + log
½H  ½acid Ka between the changes in pH and the volume of
the acid or base of known concentration added
As log 1/[H+] ¼ pH and log l/Ka ¼ pKa:
to the solution. The pH values are shown on
½salt the x-axis and the volume of added acid or base
pH ¼ pKa + log
½acid on the y axis (Fig. 2.11). During the titration of a
weak acid or base by a strong base or acid, a
The pH of a buffer system is equal to the pK of the
buffer system is formed, in which the concentra-
acid (pKa), plus the logarithm of the ratio
tion of its components changes as the titration
between the initial concentration of the salt and
proceeds. The pH changes that occur at different
the initial concentration of acid. This relationship
stages of neutralization provide data of interest
is known as the Henderson-Hasselbalch equation.
related to the buffering capacity of the system.
The buffering capacity of a buffer system var-
As an example of the titration curve of a weak
ies depending on the relative concentrations of
acid, we will analyze the neutralization of 10 mL
the acid and the salt within the system. It is max-
of 0.1 N acetic acid with a 0.1 N NaOH solution.
imal when the concentration of the acid is equal
to that of the salt. In this case, the ratio [salt]/
[acid] is equal to 1. Log1 is equal to zero, there-
fore, according to the Henderson-Hasselbalch
equation, the pH equals the pKa.
This can be conveyed as follows: the capacity
of a buffer system to minimize the pH change
produced in a medium by the addition of acids
or bases is maximal when the pH of the buffer is
equal to the pKa of the acid.

Titration curve of acids and bases

Acid-base titration is a procedure used to

determine the concentration or “titer” of an acid
(or a base) solution, by adding a known and
equivalent amount of a base (or acid) to the solu-
tion. During the titration process, a neutraliza- FIG. 2.11 Titration curve of acetic acid. The yellow area
tion reaction is produced. When the same indicates the buffer zone.
16 2. Water

Acetic acid is weakly ionized, giving H+ and CH3 COO
+ H2 O ! CH3 COOH + OH

acetate ions (CH3COO
)
It is interesting to note that the curve flattens on
CH3 COOH ! H+ + CH3 COO
either side of the titration midpoint (Fig. 2.11).
The ionization constant of the acid can be repre- Consecutive addition of base produces rela-
sented by the relationship: tively less variation in pH in an area covering
approximately one pH unit around the mid-
½H+  ½CH3
COO
 point compared to other sections of the curve.
Ka ¼
½CH3
COOH This indicates that the buffering capacity of a
buffer system is higher in the area near the mid-
The Ka of acetic acid at 25°C is 1.74  10
5 M and point and is dependent on the value of [salt]/
the pKa is 4.76. [acid]. The [salt]/[acid] ratio where the buffer
Two equilibrium conditions, the ionization of system is effective ranges from 1/10 to 10/1
acetic acid and the solvent (water, Kw ¼ [H+, and the buffer capacity is maximal when the
OH
]) are maintained in the system throughout ratio is equal to 1 (pH ¼ pKa). If one applies these
the titration. For simplicity, only the acid ioniza- values to the Henderson-Hasselbalch equation:
tion will be considered. for [salt]/[acid] ¼ 1/10:
Before NaOH is added, the pH of the medium
is equal to the pH of the 0.1 N acetic acid solu- 1
pH ¼ pKa + log ¼ pKa + log 10
1 ¼ pKa
1
tion. While adding NaOH, the following reac- 10
tion occurs:
for [salt]/[acid] ¼ 1:


CH3 COOH + NaOH ! CH3 COO + Na + H2 O +
pH ¼ pKa + log 1 ¼ pKa + 0 ¼ pKa

+
The newly added OH combines with H to for [salt]/[acid] ¼ 10/1:
form H2O molecules. The decrease in [H+] is
compensated by the ionization of the acid to 10
pH ¼ pKa + log ¼ pKa + log 101 ¼ pKa + 1
form acetate (salt). The value of Ka is maintained 1
constant. The Henderson-Hasselbalch equation This indicates that a buffer has greater buffering
can be used to calculate the pH from the buffer capacity in the pH range between 1 + pKa of the
system formed by the nonneutralized weak acid system.
and the acetate. When 50% neutralization of the
starting acid is reached, equal amounts of acid Appendix
and acetate (salt) are present in the medium.
At this point, the pH value will be equal to the Expression of concentrations
pKa (4.76) (Fig. 2.11). If the titration is continued, Concentration is the ratio between the
the remaining acid is gradually converted to ace- amount of solute and solvent in a solution. Dif-
tate. Eventually, the solution will become ferent types of expressions are used to express
completely neutralized. This occurs when the the concentration of a solute.
number of base equivalents is equal to that of Percent concentration. For the components of
the acid. The curve will show a sharp upward biological fluids it is common to indicate the
inflection, showing that the equivalence point amount of solute (weight in g, mg, μg, ng) dis-
has been reached. The pH at this point does not solved in 100 parts of solvent (by volume,
correspond to neutrality but is rather shifted to 100 mL or the equivalent, 10 dL, 1 dL ¼ 100 mL).
the alkaline side (8.72). This is due to hydrolysis Molar concentrations. A mol refers to the quan-
of the acetate, which results in increased [OH
]: tity of any element equal to the atomic mass of
 Appendix 17
that element in grams (gram atom), which con- convenient to express them in millimoles per
tains 6.022  1023 atoms (Avogadro’s number). liter (millimolar concentration or mM) or in
For example, there is the same number of atoms micromoles per liter (micromolar concentration
in 1 g of 1H as in 12 g of 12C: 6.022  1023. For any or μM).
compound, a mol is equal to its molecular To calculate the molarity of a solution when
weight in grams (gram molecule), which has its concentration is known in grams per liter,
6.022  1023 molecules. For example, 180 g of glu- the following formula is applied:
cose (C6H12O6), 60 g of urea [CO(NH2)2], and  
Concentration g=L
142 g of Na2HPO4 contain 6.022  1023 molecules. Molarity ðmol=LÞ ¼  
One mol can be defined as the quantity of Molecular weight g=mol
matter containing one Avogadro’s number of
particles (electrons, ions, or molecules). Often In general, the concentration of many substances
a 1000-fold smaller unit, the millimole (mmol), in organic liquids is given in mg per 100 mL or
is used. In certain cases, it is necessary to use dL. It is preferable to express the molarity in mil-
even smaller units, such as the micromole (μmol) limoles per liter (mM concentration); it is calcu-
equal to 10
6 mol, or the nanomole (nmol) equal lated from the concentration in mg per dL using
to 10
9 mol of substance. the formula:
Molarity is the number of moles of solute  
Concentration mg=dL  10
existing in 1 L of solution and is indicated by Molarity ðmM=LÞ ¼  
the notation M (capital m). A 1 M solution con- Molecular weight g=mol
tains 1 mol of solute dissolved in 1 L; a 0.5 M
solution, 0.5 mol/L; and a 3 M, 3 mol/L. For example, the plasma calcium concentration
Given a 0.2 M CaCl2 solution, its concentra- is 10 mg/dL or 100 mL (Ca atomic mass 40). Its
tion will be 0.2 mol of calcium chloride per liter molarity in millimoles per liter (mM) will be:
of solution. Since this salt ionizes by the 10  10
equation: Molarity ðmMÞ ¼ ¼ 2:5mM
40
CaCl2 ! Ca2+ + 2Cl
Molal concentrations. Molality refers to the moles
of solute per 1000 g of solvent. This is indicated
calcium ion concentration in the solution is 0.2 M by the symbol m (lowercase m). In this notation,
because an equal number of calcium ions as that the concentration is not influenced by tempera-
of molecules originally present in the solution ture and can be used to calculate the boiling or
(assuming full dissociation) are formed. Instead, freezing temperatures of a solution. In chemical
each mol of CaCl2 gives two chloride ions, so practice, molar solutions are most frequently
that the Cl
concentration is 0.4 M. used because volume units are preferred. In
Molarity corresponds to the ratio: this last case, it is important to mention the tem-
Moles perature at which the molar solution was
M¼ prepared.
Volume ðLÞ
Equivalents and their use for concentrations. It is
The amount of moles in a given mass of the sub- usual to express concentrations in terms of
stance is estimated by the relationship: chemical equivalents (Eq.). One equivalent gram
  or equivalent weight is the mass in grams of an
Mass g
M¼   element, ion, or compound that can displace or
Molecular weight g=mol
combine with 1 g of hydrogen or 8 g of oxygen.
Given the low concentrations of certain ions or In redox reactions, 1 Eq. is the amount of sub-
substances in biological fluids, it is sometimes stance that gains or losses 1 mol of electrons. In
18 2. Water

acid-base reactions, 1 Eq. is the amount of acid or Summary

base that liberates or accepts 1 mol of protons.
The equivalent weight is a reactive unit. Water is the most abundant component of the human
Expressing the concentration in Eq., it is possible body. Approximately 65% of the weight of an
to compare the number of chemical units that adult human is water. The function of all cells
can combine. One Eq. of an oxidizing agent and tissues depends on it.
reacts exactly with 1 Eq. of a reducer. One Eq. Water has unique properties. The particular
of acid is precisely neutralized by 1 Eq. of the molecular structure of water provides it with
base. One Eq. of Na exactly combines with physical properties, such as melting and boiling
1 Eq. of Cl or bicarbonate. temperatures, and heat of vaporization that are
The equivalent weight of an element is calcu- higher than those of other substances of similar
lated by dividing its atomic weight by the num- weight.
ber of electrons (n) that an atom gains or losses Molecular structure of water. The elements in
when the element reacts. For acids or bases, water (HdOdH) are arranged in a 104.5 degrees
the mass of 1 mol is divided by the number of angle, which gives the molecule its polar nature.
protons (n) that each molecule of acid or base The negative charge is displaced toward the
yields or accepts. In the case of an ion, the mole O atom and the positive charges to the H atoms.
weight is divided by the number of electric Hydrogen bond. The charge distribution within
charges (n) it contains. Thus, one equivalent of the water molecule allows the formation of
the following substances will be: sodium, hydrogen bonds between different water mole-
23/1 ¼ 23 g; chloride, 35.5/1 ¼ 35.5 g; calcium, cules (the positive charge of an H in a water mol-
40/2 ¼ 20 g; bicarbonate (HCO
3 ), 61/1 ¼ 61 g; ecule is attracted by the negative charge of
and phosphate (PO3
4 ), 95/3 ¼ 31.6 g. another). In this manner, water can form poly-
As the concentrations of electrolytes in body meric complexes with the formula (H2O)n. These
fluids are low, it is customary to express them complexes are more common in solid (ice) or liq-
in milliequivalents (mEq.) per liter. A mEq. is uid water, than in water vapor. Each water mol-
one-thousandth of an equivalent. ecule can form hydrogen bonds with another
Frequently, the concentration of a specific ion four molecules. In ice, a regular crystal lattice
in biological fluids is given in mg per dL, and is formed, which has fixed distances between
this expression must be converted into millie- molecules. In liquid water, the H bonds easily
quivalents per liter. The conversion formula form and break apart, giving the molecules the
used in this case is: freedom to move closer; this explains why liquid
  n water is denser than ice.
mg=dL  10  ¼ mEq:=L Polarity of water molecules influences their inter-
Atomic mass
action with other substances. The electrostatic
Examples: sodium concentration in plasma is attractions of ionic and polar nonionic com-
322 mg/dL. Expressed in mEq./L, the concen- pounds favor their interactions with water. They
tration will be: are hydrophilic substances that can form stable
1 water solutions. Nonpolar compounds are
322  10  ¼ 140mEq:=L
23 hydrophobic and do not dissolve in water.
The plasma calcium concentration is 10 mg/dL. Amphipathic substances which exhibit hydro-
Expressed in mEq/L as: philic and hydrophobic groups in the same mol-
ecule (phospholipids and soaps) can form
2
10  10  ¼ 5mEq:=L micelles in water. These molecules have their
40 polar groups oriented toward the polar aqueous
 Further reading 19
medium and the hydrophobic groups toward which functions as a strong electrolyte. The pH
the interior of the micelle, where they are mutu- of the buffer solutions can be calculated by
ally attracted by hydrophobic interactions. knowing the acid dissociation constant (Ka)
Water is an electrolyte. Water weakly dissoci- and the initial concentrations of the acid and
ates into hydrogen and hydroxide ions. In pure the salt. The relationship between these values
water at 25°C, the hydrogen ion concentration is expressed in the Henderson-Hasselbalch
[H+] and the hydroxyl ion concentration equation:
[OH
] are the same, 0.0000001 M or 10
7 M.
½salt
Water ion product (Kw). The product [H+]  pH ¼ pKa + log
[OH
] is a constant. Its value at 25°C is 10
7  ½acid
10
7 ¼ 10
14. This remains constant in both pure Buffers display their highest buffering capacity
water and aqueous solutions. For this reason, at a pH range between 1 + pKa.
when a substance that increases [H+] or [OH
] Titration curve of weak acids. This is obtained by
is added to aqueous solutions, a concomitant plotting the changes in pH produced by neutral-
decrease of [OH
] or [H+] immediately occurs, ization of the acid with a strong base against the
trying to maintain the value of the product volume of the base used. The concentrations of
[H+]  [OH
] constant at 10
14. the components of the buffer system formed dur-
Acids and bases. When [H+] is equal to [OH
], ing the titration changes as the base are added.
the solution is considered neutral. Any sub- The buffering capacity of the system is maximal
stance dissolved in water, which increases when the pH of the solution equals to the pKa.
[H+] is an acid; and any that decreases [H+], is
a base or alkali. According to Brønsted and
Lowry, acids are compounds that yield H+ ions Further reading
or protons to the solution and bases are the com- Chaplin, M., 2006. Do we underestimate the role of water in
pounds that can accept protons from the cell biology? Nat. Rev. Mol. Cell Biol. 7, 861–866.
medium. According to their degree of ioniza- Hezavehei, M., Sharafi, M., Kouchesfahani, H.M., Henkel, R.,
Agarwal, A., Esmaeili, V., Shahverdi, A., 2018. Sperm
tion, acids and bases can be strong or weak. cryopreservation: a review on current molecular cryobi-
pH. To simplify the expression of [H+] in a ology and advanced approaches. Reprod. BioMed.
solution, the pH notation is used. The pH corre- Online 37, 327–339.
sponds to the logarithm of the reciprocal of the Mazur, P., Paredes, E., 2016. Roles of intracellular ice forma-
[H+] or the negative logarithm of [H+]. For pure tion, vitrification of cell water, and recrystallization of
intracellular ice on the survival of mouse embryos and
water at 25°C, [H+] ¼ 10
7 M, the pH ¼
oocytes. Reprod. Fertil. Dev. 28 (8), 1088.
log10
7 ¼ 7. At this temperature, acids have a Nicolls, P., 2000. Introduction to the biology of the water mol-
pH below 7 and alkalis or bases, above 7. ecule. Cell. Mol. Life Sci. 57, 987–992.
Buffers. These are systems that minimize the Paladini, A.C., 1983. Agua. In: Torres, H.N., Carminatti, H.,
changes in [H+] produced by the addition of Cardini, C.E. (Eds.), Bioquı́mica General. El Ateneo, Bue-
nos Aires, pp. 81–90.
acids or bases to a solution. A buffer solution Stewart, P.A., 1981. How to Understand Acid–Base:
is generally constituted by a mixture of a weak A Quantitative Acid–Base Primer for Biology and Medi-
electrolyte (commonly a weak acid) and its salt, cine. Edward Arnold Ltd., London.
This page intentionally left blank
 C H A P T E R

3
Proteins

Proteins are molecules of immense importance All proteins contain carbon, hydrogen,
for living organisms. They are the most abundant oxygen, and nitrogen and most also have sul-
organic compounds in vertebrates, accounting for fur. Although there are slight variations, the
approximately 50% of their tissue dry weight. nitrogen content in proteins is approximately
Virtually all biological processes depend on the 16% of their total molecular mass. Thus,
function of proteins. The following are only a 6.25 g of protein contains 1 g of nitrogen. The
few of the many examples of different protein value 6.25 is used as a factor to estimate the
types and the variety of roles that they perform: amount of protein in a sample with known
enzymes catalyze most chemical reactions in the N content.
body; hormones regulate many cellular activities;
hemoglobin and transport proteins carry a series
of substances in blood; antibodies defend the body Proteins are macromolecules formed
against the attack of foreign agents; receptors trig- by amino acids
ger specific responses in cells; actin and myosin
allow muscle contraction, and collagen form the Proteins are large molecules (macromole-
highly resistant fibers of connective tissue. cules), polymers of structural units called amino
Major advances have been made in under- acids. A total of 20 different amino acids exist in
standing the structure and function of proteins proteins and hundreds to thousands of these
over the last several years. This has helped to amino acids are attached in long chains to form
explain the molecular basis underlying many a protein. Amino acids can be released from pro-
biological processes. One of the most challeng- teins by hydrolysis (Hydrolysis is the cleavage
ing problems in protein research is the isolation of a covalent bond by the addition of water in
and purification of proteins from the extremely adequate conditions).
complex mixture of molecules that compose the Due to their large size, proteins obligatorily
living matter. The development of research tools form colloids when they are dispersed in a suit-
for the separation of proteins in a pure form, and able solvent. This property characteristically dis-
their crystallization, allowed understanding the tinguishes proteins from solutions containing
structure and function of many proteins. small size molecules.

Medical Biochemistry 21 Copyright # 2022 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/B978-0-323-91599-1.00004-3
22 3. Proteins

Since amino acids are the “building blocks” For example, in the amino acid alanine:
for proteins, their structure and properties will
be considered first.

Amino acids The asymmetric carbon, in red, is bound to four

different atomic groups (dH, dCH3, dNH2,
Amino acids are compounds that have an and dCOOH).
acid or carboxyl (dCOOH) group, and a basic The groups attached to the central asymmet-
or amine (dNH2) group, both of which are ric carbon can be spatially arranged in two dif-
attached to an alpha carbon (αC) of an organic ferent ways. This results in two molecules,
acid (the α carbon is that located next to the car- each of which is the mirror image of the other.
boxyl group). That is why amino acids are called As their relationship is analogous to that of the
a-amino acids, and their general formula is: left and right hands, these compounds are called
chiral compounds (from the Greek chiros: hand).
They are also known as optical isomers, enantio-
morphs, or enantiomers. Fig. 3.1 shows the two
optical isomers of alanine.
R corresponds to the side chain, which varies for Many of the chemical and physical properties
each of the 20 amino acids obtained by protein of enantiomers are identical, except for the
ability of these compounds to deflect polarized
hydrolysis.
light.
According to the general formula, the αC of
Polarized light. A beam of ordinary light con-
all amino acids (except glycine, with H as a side
sists of waves vibrating in all planes intersecting
chain) is bound to four different functional
the axis of beam propagation (Fig. 3.2). In con-
groups. This allows for the existence of two opti-
cal isomers with different spatial arrangements trast, polarized light consists of waves that
for each amino acid. vibrate in a single plane. Polarized light can be
obtained by passing a beam of ordinary light
through a Nicol prism (device constructed with
Optical isomerism calcite crystals), or through the synthetic mate-
rial called Polaroid.
Isomers refer to compounds that have the
Optical activity. If a beam of polarized light
same molecular formula but are structurally dif-
passes through a solution of a chiral compound,
ferent. When isomers only differ in the spatial
arrangement of their atoms, they constitute spa-
tial isomers, or stereoisomers, a group that also
includes the optical isomers. According to the
tetrahedral configuration, the four bonds of
the carbon atom are equivalent and oriented to
the vertices of the tetrahedron. When each car-
bon valence is bound to different elements or
atomic groups, the molecule becomes asymmet-
ric and the carbon involved is known as asym-
metric carbon. FIG. 3.1 Alanine enantiomers.
 Amino acids 23
The angle of rotation of the polarized light
plane depends on several conditions, including
temperature, light wavelength, concentration,
and thickness of the solution that is traversed
by light. Moreover, if the conditions mentioned
above are maintained constant, an optically
active compound rotates the polarized light to
a degree that is unique for that substance
FIG. 3.2 (A) Graphical representation of an ordinary light
beam. The circle on the right represents the ideal cross section (specific rotation). Optical isomers of the same
of the beam. Only some of the infinite planes of vibration that compound deviate the polarized light in the
intersect at the axis of propagation of the beam are repre- same manner; however, while one turns polar-
sented. (B) Polarized light; the wave vibrations occupy only ized light to the right, the other turns it to
one plane.
the left.
Notation. Two optical isomers can be distin-
guished by the configuration of the four substit-
uents around the chiral or asymmetric carbon,
which can be determined using X-ray diffrac-
tion. The compound that is taken as a reference
is glyceraldehyde. Both optical isomers of this
substance, one levorotatory and the other dex-
trorotatory, are designated L and D respectively.
Their chemical formulas are the following:

FIG. 3.3 Rotation of polarized light. The plane of light

vibration, after passing through a tube containing a solution
of an optically active substance is deviated from its original
position (in this case, a clockwise deviation is shown).

the plane of light vibration is rotated on its axis

(Fig. 3.3). It is said that the substance is optically The L-isomer is represented by placing the
active. By convention, when the rotation is in the hydroxyl group to the left of the asymmetric car-
clockwise direction, it is considered to be right or bon and the D-isomer, with the carboxyl group to
positive (+); when the rotation takes place in the the right.
counter-clockwise direction, it is considered to By convention, all compounds that have a
be left or negative (
). similar configuration to that of L-glyceraldehyde,
Compounds that deflect the plane of vibra- are called L, even if they are not levorotatory. Sim-
tion of polarized light to the right are called dex- ilarly, compounds that are analogous to the spa-
trorotatory and those that rotate it to the left, are tial arrangement of D-glyceraldehyde are called
D, although they may not be dextrorotatory.
known as levorotatory.
The degree of rotation can be estimated by an Thus, the D and L denomination denote the con-
instrument called polarimeter, which measures figuration concerning the αC and does not
the angle of rotation of the polarized light plane always correspond to the dextrorotatory and
when it passes through a solution of an optically levorotatory activities of the compound. For this
active substance. reason, the direction of polarized light rotation
24 3. Proteins

must be indicated with a (+) or (
) sign after the TABLE 3.1 Nomenclature of amino acids.
letter L or D. L-Alanine, for example, has a specific
rotation of +1.8 degrees and its notation is there- Amino acid Three letter One letter
name notation notation
fore, L(+)-alanine.
Alanine Ala A
Arginine Arg R
Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C
To avoid ambiguities with the D and L designa- Glutamic acid Glu E
tion, another system called RS has been pro-
posed. However, the notation DL continues to Glutamine Gln Q
be the one widely used. All amino acids, except Glycine Gly G
glycine, offer D and L isomers. Isoleucine and Histidine His H
threonine, amino acids that have a second asym-
metric carbon in addition to the αC, can produce Isoleucine Ile I
four isomers. Only one of these isomers will par- Leucine Leu L
ticipate in the formation of proteins. Lysine Lys K
Cells can distinguish stereoisomers with
great efficacy. Only amino acids with the Methionine Met M
L-configuration are incorporated into the pro- Phenylalanine Phe F
teins of the human body. We will almost exclu- Proline Pro P
sively refer to them in the following pages. Thus,
when no letter is included to the name, it is Serine Ser S
understood that we are referring to L-amino Threonine Thr T
acids. Tryptophan Trp W
Tyrosine Tyr Y
Classification of amino acids
Valine Val V
Most of the 20 α-amino acids contain one
acidic carboxyl group and one basic amine
group, bound to the αC, which gives them a neu-
tral net charge. Two amino acids have an addi- Next, amino acids will be presented classifying
tional carboxyl group that gives the molecule them according to the characteristics of their
acidic characteristics. Others, in contrast, have side chains. The chemical formula is shown with
additional basic groups. Two amino acids con- their side chains highlighted in red.
tain sulfur. Finally, proline is the only amino
acid in which the carbon adjacent to the carboxyl Neutral amino acids with a nonpolar
group forms part of a cyclic five-membered ring. aliphatic chain
Each amino acid is indicated by abbreviated Glycine only has one hydrogen in its side
notations, one of which uses three letters, and chain and the polar groups, carboxyl, and
another uses only one letter. A list of the amino amine, have a predominant role in the molecule.
acids with their notation is shown in Table 3.1. Alanine, with a methyl group side chain, is more
 Amino acids 25
soluble in water than those containing hydro- Aromatic neutral amino acids
phobic chains. Valine, leucine, and isoleucine have Phenylalanine, which contains a benzene ring,
nonpolar branched chains. and tryptophan, with an indole heterocyclic side
chain, are both markedly nonpolar and hydro-
phobic. Tyrosine has a phenolic hydroxyl that
adds to the polarity of this amino acid. At pH
values above 10, tyrosine releases a proton and
becomes negatively charged.

These three amino acids with aromatic side chains

strongly absorb light in the ultraviolet range of the
light spectrum (280 nm). This property is used to
Neutral aliphatic amino acids with a detect the presence of proteins in a sample.
nonionizable polar chain
Serine and threonine contain a hydroxyl func- Amino acids containing sulfur
tional group in their side chains, which gives Cysteine contains a sulfhydryl group (dSH)
these amino acids polar characteristics. that is slightly polar. At a pH 9, it will release a
proton. Methionine has a nonpolar side chain.
26 3. Proteins

Acidic (dicarboxylic) amino acids

Aspartic acid and glutamic acid are amino acids
that have an additional carboxyl group that
can release a proton and acquires a negative
charge at the pH of body fluids. Often, these
amino acids are designated with the name of
their ionized form, aspartate, and glutamate,
respectively.

The side chain of histidine is the heterocyclic

imidazole side chain. One of the nitrogen atoms
in imidazole can acquire a positive charge. The
side chain of histidine has an ionization pKa
Asparagine and glutamine are derivatives of value of around 6.0, so it can act as a base. The
aspartic and glutamic acid, respectively; they basic amino acids, lysine, and histidine are
possess an amide functional group in the highly polar.
carbon distal from the α carbon. Unlike their
acidic analogs, the side chains of asparagine
and glutamine have no electric charge; they are
polar.

The protein collagen contains hydroxylysine,

which is a lysine derivative that has a hydroxyl
group at carbon 5 (chain carbons are counted
starting from the carboxyl group).

Proline
In the amino acid proline, the α carbon and
Basic amino acids the nitrogen bound to it are included in a pyrro-
Lysine has an additional amine functional group lidine cycle. This chemical ring (not aromatic)
and arginine has a guanidine group, both of gives the amino acid an aliphatic character. Ali-
which can accept protons. At physiological phatic compounds are those composed of
pH, the residues in these amino acids display straight chained, branched, or cyclic compounds
a positive electrical charge. and can be saturated or unsaturated. In contrast,
 Amino acids 27
aromatic compounds contain double bonds in a Other biologically important amino acids
benzene or heterocyclic ring. exist as free compounds or are part of nonpro-
tein molecules. These include α-alanine, D-ala-
nine, sarcosine, α-aminobutyric acid,
D-glutamic acid, ornithine, homoserine, tyro-
sine, citrulline, and homocysteine.

Some authors consider that the nitrogen of pro-

line forms an imino function (]NH) and, there-
fore, they call proline an imino acid rather than
an amino acid. Having the α carbon and N in the
ring gives the proline molecule greater stiffness
than other amino acids. Some proteins contain a
hydroxylated derivative of proline, the
hydroxyproline.

Other amino acids

Some of the amino acids presented above can
be modified by the covalent addition of different
chemical groups. This includes, for example,
phosphoserine, α-carboxyglutamic acid, and
the already mentioned 4-hydroxyproline and
5-hydroxylysine.

Much less common than the amino acids men-

tioned earlier are selenomethionine and seleno-
cysteine, in which the sulfur of cysteine and
methionine is replaced by selenium. Approxi-
mately 15 proteins (selenoproteins) possessing
these selenoamino acids have been described.
Selenocysteine is preferably found in animal
proteins, while selenomethionine is mainly
found in plant proteins.
28 3. Proteins

General properties of amino acids usual in biological media. In the crystalline state
or in aqueous solutions, amino acids ionize, giv-
Based on the properties of each amino acid ing rise to both positive and negative charges on
chain, one can predict their behavior. The sulf- the same molecule. For this reason, amino acids
hydryl group of cysteine is highly reactive and are considered dipolar ions, or ampholytes. The
easily combines with other sulfhydryl groups German word zwitterion (hybrid ion) is also used
to form disulfide bonds (dSdSd). Two cyste- to describe these types of polar molecules.
ines linked by this type of covalent bond form Therefore, it is more correct to represent the
a compound known as cystine. α-amino acids as dipolar ions:

The additional carboxyl group of aspartic and Where the ionized groups are shown in red.
glutamic acids not only give them an acidic char- The electrical charge of an amino acid
acter, but also the ability to interact with basic depends on the pH of the medium in which it
substances to form salt-like bonds. The basic dia- is dissolved. Increases in the hydrogen ion con-
minated amino acids can also establish electro- centration in the medium diminish the dipolar-
static bonds with acid substances. ity of the amino acid because the carboxylate
Amino acids can be grouped based on the (dCOO
) groups accept protons and act as
polarity of their side chains: bases. The amino acid then becomes a cation.
Polar amino acids include glycine, serine,
threonine, cysteine, tyrosine, aspartic acid, glu-
tamic acid, asparagine, glutamine, lysine, histi-
dine, and arginine.
Nonpolar amino acids are alanine, valine, leu-
cine, isoleucine, methionine, phenylalanine, In contrast, when the amino acid is in an alkaline
tryptophan and proline. medium, the concentration of H+ decreases and
OH
increases; the dNH+3 groups act as an acid,
releasing H+. The amino group becomes nega-
tively charged (it is an anion).
Acid-base properties of amino acids
The existence of both an acidic and a basic
group within the same molecule gives amino
acids particular electrical properties. The car-
boxyl group behaves as an acid or proton donor:
▬COOH ! ▬COO
+ H+ There is a pH value, characteristic for each
amino acid, in which the ionization of positive
The amine group accepts protons and acts as a and negative charges is equal and, therefore,
base: the electric charge of the whole amino acid is
▬NH2 + H+ ! ▬NH+3 zero. This pH value is called the isoelectric point
(pHi or pI). At the pHi, an amino acid in a solu-
In the formulas shown earlier, the amino acids tion subjected to an electrical field will not
are in a nonionized state, a situation that is not migrate toward either electrode.
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vaativan. Senvuoksi oli minusta myös sopimatonta, vaikken ollut
nähnyt häntä sitten viime joulun, osoittaa minkäänlaista
hämmästystä tapaamisemme ajan ja paikan johdosta; ystävällisesti
vaikka näköjäni välinpitämättömänä vastasin minä hänen
tervehdykseensä ja olin juuri aikeissa ruveta keskustelemaan hänen
kanssaan, niinkuin maailmanmiesten sopii, joita ei tilapäinen
tapaaminen Austraaliassakaan hämmästytä, kun hän tehden
torjuvan kädenliikkeen lyhyesti huomautti: "Suokaa anteeksi, arvoisa
ystäväni, mutta minun aikani on rajoitettu ja minä olen tullut tänne
ainoastaan siinä tarkoituksessa, että saan kertoa Teille erään sangen
kummallisen tarinan, luonnollisesti edellyttäen, että Te olette
taipuvainen sitä kuuntelemaan."

Minä kuuntelin tätä puhetta ihmetyksellä, mutta siitä huolimatta

selitin minä hänelle olevani heti valmis häntä kuulemaan, kuitenkaan
en voinut olla kasvojeni ilmeillä ilmaisematta hämmästystäni siitä,
että tohtori Wehwald ei ollut tullut tapaamaan minua kahvilassa, ja
miten hänen oli onnistunut löytää minut yöllä täällä kaupungin
puistossa, ja vihdoin, miksi juuri minä sain kunnian kuulla hänen
kertomustansa.

"Vastauksen kahteen ensimäiseen kysymykseenne", sanoi hän

oudoksuttavan jyrkästi, "saatte Te kertomukseni kuluessa itsestään.
Mutta syy, miksi minä juuri Teidät valitsin, arvoisa ystäväni (hän ei
minua muulla nimellä puhutellutkaan) on se, että Te tietääkseni
harjoitatte myös kirjailija-tointa, jonka vuoksi minä uskallan toivoa,
että Te julkaisette tämän minun omituisen, vaikkakin jonkunverran
katkonaisen kertomukseni sopivasti muodostettuna."

Minä estelin vaatimattomasti, jolloin tohtori Wehwald nyrpisteli

nenäänsä kummallisesti ja alkoi ilman minkäänlaista johdatusta:
"Kertomukseni sankarittaren nimi on Redegonda. Hän oli parooni T:n
puoliso, joka oli ratsumestarina meidän pikkukaupunkiimme
sijoitetussa rakuunarykmentissä." (Hän mainitsi tosiaankin vaan
nämä alkukirjaimet, vaikka minulle ei tuon pienen kaupungin nimi,
eikä liioin ratsumestarinkaan nimi ja rykmentin numero olleet, syistä,
jotka pian selviävät, mikään salaisuus.) "Redegonda", jatkoi tohtori
Wehwald kertomustaan, "oli harvinaisen kaunis nainen ja minä
rakastuin häneen, niinkuin sitä tavallisesti sanotaan, heti ensi
hetkessä. Mutta valitettavasti ei minulla ollut mitään tilaisuutta
päästä hänen kanssaan persoonallisesti tutuksi, sillä upseerit eivät
seurustelleet juuri nimeksikään sivilihenkilöitten kanssa ja kohtelivat
meitä valtion virkamiehiäkin melkein loukkaavan ylimielisesti. Näin
ollen näin minä Redegondan aina vain kaukaa; näin hänet yksin tai
miehensä kanssa, usein myös toisten upseerien ja heidän naistensa
kanssa kadulla kävelemässä, joskus näin hänet myös Päätorin
varrella olevan asuntonsa ikkunassa, tai iltasin ajavan huonoissa
vaunuissa kaupungin pieneen teatteriin, missä minulla sitten oli onni
permannolta katsella häntä omassa aitiossaan, jonne nuoret upseerit
väliajoilla mielellään kiiruhtivat. Joskus tuntui minusta, että hän
suvaitsi huomata minut. Mutta hänen katseensa viivähti minussa
aina vaan niin hetkisen, etten voinut siitä mitään enempää päättää.
Minä olin jo heittänyt kaiken toivonkin koskaan saada ilmaista
hänelle sydämeni palavia tunteita, kun hän eräänä ihanana
syksypäivänä aamupäivällä tuli vastaani aivan odottamatta pienessä
puistomaisessa metsässä, joka kaupungin itätorilta ulottuu kauvas
maalle päin. Huomaamattomasti hymyillen kulki hän ohitseni, ehken
hän ei minua ollenkaan huomannutkaan, ja hävisi taas pian
keltaisten lehvien taakse. Minä olin antanut hänen kulkea ohitseni
ollenkaan ajattelematta sitä mahdollisuutta, että olisin voinut häntä
tervehtää tai puhutellakin; ja nytkin, kun hän jo oli hävinnyt, en
katunut ollenkaan, etten sitä tehnyt, sillä eihän siitä kuitenkaan olisi
voinut olla mitään tulosta. Mutta sitten tapahtui jotain kummallista:
minä tunsin nimittäin itseni äkkiä pakotetuksi kuvittelemaan, mitä
olisi tapahtunut, jos minulla olisi ollut rohkeutta asettua hänen
tielleen ja puhutella häntä. Ja mielikuvituksissani näin minä, kuinka
Redegonda ollenkaan minuun vihastumatta päinvastoin ihan
peittelemättä ilmaisi tyytyväisyytensä rohkean tekoni johdosta, ja
vilkkaasti keskustellessamme valitti elämänsä tyhjyyttä ja
seurapiirinsä ala-arvoisuutta, ja lopuksi lausui ilonsa siitä, että oli
minusta löytänyt ymmärtävän, samoin ajattelevan toverin. Ja hänen
jäähyväiskatseensa, joka minut kohtasi oli niin lupaava, että minusta,
joka kaiken tämän elin vain mielikuvituksissani, samana iltana
nähdessäni hänet jälleen aitiossaan tuntui kuin meillä kahdella olisi
yhteinen ihana salaisuus. Teitä ei kai ihmetytä, arvoisa ystävä, että
minä, joka nyt olin saanut mielikuvitusvoimastani niin erinomaisen
todistuksen, yhä edelleen jatkoin samanlaisia tapautumiskohtauksia,
ja että ne kerta kerralta tulivat yhä ystävällisemmiksi,
tutunomaisemmiksi, jopa ihan sydämellisiksi, kunnes eräänä ihanana
päivänä lehdettömien oksien alla ihailemani nainen vaipui ikävöivään
syliini. Minä jatkoin yhä edelleen tätä huumaavaa mielettömyyttäni,
eikä kauvan enää kestänytkään, ennenkuin Redegonda tuli minua
tapaamaan pieneen, kaupungin syrjässä olevaan asuntooni, ja minä
sain nauttia sellaista onnea, jota köyhä todellisuus ei koskaan olisi
voinut niin huumaavana minulle tarjota. Vaarojakaan ei puuttunut,
jotka seikkailuamme höystivät. Niinpä tapahtui kerran talvella, että
ratsumestari ratsasti ohitsemme ajaessamme yöllä reellä maantiellä
turkkiin käärittyinä; ja silloin jo aavistin sen julman kohtalon, joka
sitten pian toteutuikin. Ensimäisinä kevätpäivinä saatiin kaupungissa
tietää, että se rakuunarykmentti, johon Redegondan mieskin kuului,
tulisi siirrettäväksi Galiziaan. Minun, ei, — meidän epätoivomme oli
rajaton. Kaikkea, mitä rakastavaiset sellaisissa tavattomissa oloissa
tavallisesti harkitsevat, mietittiin: yhteistä pakoa, yhteistä kuolemaa,
tuskaista alistumista välttämättömyyteen. Viimeinen ilta tuli
kuitenkin, ilman että mitään varmaa päätöstä oli tehty. Minä odotin
Redegondaa kukilla koristetussa huoneessani. Kaikkien
mahdollisuuksien varalta oli matka-arkkuni pakattu, revolverini
ladattuna —, ja jäähyväiskirjeet kirjoitettuina. Tämä kaikki on totta,
arvoisa ystäväni. Sillä niin täydellisesti olin minä joutunut
mielettömyyteni valtaan, että minä en pitänyt rakastettuni
ilmestymistä sinä iltana, joka oli viimeinen ennen rykmentin lähtöä,
ainoastaan mahdollisena, vaan minä suorastaan odotinkin sitä. Minä
en kuitenkaan voinut, niinkuin tavallisesti, loihtia hänen haahmoansa
luokseni, enkä mielikuvituksessani syleillä tuota taivaallista olentoa;
ei, minusta tuntui kuin jokin odottamaton, ehken jokin kauhea,
pidättäisi häntä kotonaan; satakertaa menin minä ovelle
kuuntelemaan kuuluisiko portaista askeleita, satakertaa katsoin
ikkunasta nähdäkseni Redegondan lähestyvän kadulla; niin,
kärsimättömyydessäni olin jo juosta pois Redegondaa etsimään,
hakemaan hänet luokseni, rakastavan ja rakastetun oikeudella
uhkamielisenä vaatimaan häntä mieheltään, — kunnes vihdoin
vaivuin leposohvalleni, aivankuin kuumeessa. Silloin äkkiä, oli jo
melkein puoliyö, kuului ulkoa ovikellon soitto. Minä tunsin sydämeni
pysähtyvän. Sillä kellon kilinä, ymmärrättehän kai, ei ollut enää
mitään mielikuvitusta. Se soi toisen ja kolmannen kerran ja herätti
minut jyrkästi ja auttamattomasti täyteen todellisuuteen. Mutta
samassa hetkessä, kun minä huomasin, että seikkailuni tähän iltaan
asti olikin ollut vaan ihmeellistä unennäköä, tunsin minä rohkeimman
toivon heräävän sielussani: sen nim. että Redegonda, toiveitteni
voiman järkyttämänä ja pakottamana omassa persoonassaan seisoisi
kynnykselläni, ja että minä seuraavassa hetkessä saisin hänet ilmi
elävänä sulkea syliini. Tässä ihanassa odotuksessa menin minä
ovelle ja aukaisin. Mutta se ei ollutkaan Redegonda, joka edessäni
seisoi, se oli Redegondan puoliso; hän itse, niin elävänä ja
todellisena kuin Te nyt tässä penkillä minun vastapäätäni, tuijottaen
jäykkänä kasvoihini. Minä en voinut tietysti muuta kuin päästää
hänet sisään, ja pyytää häntä istumaan. Mutta hän jäi seisomaan ja
sanoi selittämättömän ivallisesti: 'Te odotatte Redegondaa. Mutta,
ikävä kyllä, hän ei voi tulla. Hän on nimittäin kuollut.' 'Kuollut', toistin
minä, ja koko maailma pysähtyi. Ratsumestari puhui häiriintymättä
edelleen: 'Noin tunti sitten löysin minä hänet kirjoituspöytänsä
äärestä istumasta, tämä pieni kirja edessään, jonka minä selvyyden
vuoksi otin heti mukaani. Se oli luultavasti pelko, joka hänet tappoi,
kun minä niin odottamatta astuin hänen huoneeseensa. Nämä rivit
tässä ovat viimeiset, jotka hän kirjoitti. Olkaa hyvä!' Hän ojensi
minulle avonaisen, sinipunervaisen nahkakantisen kirjan ja minä luin
seuraavat sanat: 'Nyt jätän minä kotini ainiaaksi, rakastettuni
odottaa.' Minä vaan nyökäytin päätäni vahvikkeeksi. 'Te olette tietysti
arvannut', jatkoi ratsumestari, 'että Teillä on kädessänne
Redegondan päiväkirja. Tahdotteko ehken olla niin hyvä ja silmäillä
sitä, tullaksenne vakuutetuksi, että kaikki kieltäminen on turhaa.'
Minä silmäilin, ei, minä luin. Minä luin melkein tunnin verran
kirjoituspöytääni nojaten, ratsumestarin istuessa sillä aikaa
liikkumatonna leposohvallani; minä luin meidän rakkautemme koko
kertomuksen, suloisen, ihmeellisen kertomuksen, — kaikkine
yksityiskohtineen; siitä syksyaamusta alkaen jolloin minä metsässä
ensi kerran olin Redegondaa puhutellut, minä luin ensimäisestä
suudelmastamme, kävelyretkistämme, huviretkistämme maalle,
ihanista onnenhetkistämme kukkakoristeisessa huoneessani, meidän
pako- ja kuolemantuumistamme, onnestamme ja epätoivostamme.
Kaikki oli tähän kirjaan kirjoitettu, kaikki — mitä ei koskaan
todellisuudessa ollut tapahtunut, — mutta kuitenkin kaikki juuri
samalla tavalla kuin sen mielikuvituksessani olin elänyt. Enkä minä
pitänyt sitä niin selittämättömänä kuin Te, arvoisa ystävä, näytätte
sitä ilmeisesti pitävän. Sillä minä aavistin yhtäkkiä, että Redegonda
oli rakastanut minua yhtä paljon kuin minä häntä, ja että hän
senkautta oli saanut salaperäisen voiman eläytyä kaikkiin minun
mielikuvitukseni eläymyksiin. Ja koska hän naisena oli lähempänä
kuin minä elämän alkutilaa, missä toivo ja täyttymys ovat sama asia,
niin oli hän luultavasti ollut täysin vakuutettu siitä, että hän oli
todella elänyt kaiken sen, mikä nyt hänen sinipunervaiseen kirjaansa
oli kirjoitettu. Mutta minusta oli vielä sekin mahdollista: että tämä
koko päiväkirja ei ollut mitään muuta kuin harkittu kosto minulle,
kosto siitä päättämättömyydestä, etten ollut tehnyt minun, meidän
unelmiamme todellisuudeksi; niin, ja että hänen äkillinen
kuolemansa tapahtui hänen omasta tahdostaan ja että hän
tahallisesti antoi tuon petollisen päiväkirjan sillä tavoin joutua
petetyn miehensä käsiin. Mutta minulla ei ollut aikaa kauvan
selvitellä näitä kysymyksiä, sillä ratsumestarille oli vaan yksi ainoa
luonnollinen selitys olemassa; minä tein siis sen, mitä tilanne vaati ja
ilmoitin tavanmukaisin kääntein olevani hänen käytettävissään."

"Yrittämättä —"

"Kieltääkö?!" keskeytti tohtori Wehwald minut jyrkästi. "Oh! Vaikka

sellaisella yrityksellä olisi ollut joitakin onnistumisen edellytyksiäkin
olisi se minusta näyttänyt raukkamaiselta. Sillä minä tunsin itseni
täysin vastuunalaiseksi kaikista seikkailuni seurauksista, seikkailun,
jonka olin tahtonut kokea, mutta jonka kokemiseen minä olin ollut
vaan liian arka. — 'Minä tahtoisin', sanoi ratsumestari, 'päättää
asiamme ennenkuin Redegondan kuolema tulee tunnetuksi. Kello on
nyt yksi yöllä, kello kolme kokoontuvat todistajamme ja kello viisi
pitää kaiken olla suoritettuna.' Minä nyökkäsin taasen myöntymyksen
merkiksi. Ratsumestari poistui kylmästi tervehtien. Minä järjestin
paperini, poistuin kotoani, ja hain kaksi tuttavaa herraa sängyistään
— toinen heistä oli kreivi — kerroin heille ainoastaan, mitä
tarpeellista oli, saadakseni heidät järjestämään asian nopeasti, ja
kävelin sitten edes takaisin Päätorilla pimeitten ikkunoiden edessä,
joitten takana tiesin Redegondan ruumiin makaavan ja tunsin
varmasti käyväni kohtaloni täyttymistä kohden. Kello viisi varhain
aamulla seisoimme me vastatusten, ratsumestari ja minä, pistoolit
kädessä pienessä metsässä aivan lähellä sitä paikkaa, missä minä
ensi kerran olisin voinut puhutella Redegondaa."

"Ja Te tapoitte hänet?"

"En. Minun kuulani lensi aivan hänen ohimonsa ohi. Mutta hän
osasi minua keskelle sydäntä. Ja minä kuolin siihen paikkaan,
niinkuin on tapana sanoa."

"Ah!" huusin minä voihkien, neuvottomana katsahtaen ihmeellistä

naapuriani. Mutta katseeni ei löytänyt häntä enää. Sillä tohtori
Wehwald ei enää istunutkaan penkin nurkassa. Niin, minulla on syitä
luulla, ettei hän ylipäänsä koskaan ollut siinä istunut. Sitävastoin
muistin minä heti, että eilen illalla oli kahvilassa puhuttu paljon
eräästä kaksintaistelusta, jossa eräs ratsumestari Tellerheim oli
ampunut ystävämme, tohtori Wehwaldin. Se seikka, että rouva
Redegonda vielä samana päivänä oli hävinnyt jäljettömiin erään
rykmentin luutnantin kanssa, herätti pienessä seurapiirissä,
vallitsevasta vakavasta mielialasta huolimatta, jonkinlaista
surumielistä hilpeyttä, ja joku lausui sen arvelun, että tohtori
Wehwald, jota me aina olimme pitäneet täsmällisen, hienon ja
ylhäisen käyttäytymisen esikuvana, aivan luonteensa mukaisesti,
puoleksi tahtoen, puoleksi tahtomattaan, oli joutunut kuolemaan
toisen, onnellisemman edestä.

Mitä taas tohtori Wehwaldin ilmestymiseen kaupungin puiston

penkillä tulee, niin olisi sen ihmeellisyys sanottavasti voittanut, jos se
olisi tapahtunut ennen hänen ritarillista kuolemaansa. Enkä minä
tahdo salata, että aluksi hiukan ajattelin tehdä kertomukseeni tämän
vähäpätöisen muutoksen kohottaakseni siten sen vaikuttavaisuutta.
Vähän aikaa mietittyäni luovuin kuitenkin tästä aikeesta peläten, että
minua syytettäisiin, poikkeamalla totuudesta, antaneeni vettä
myllyyn mystiikalle, spiritismille ja muille sellaisille vaarallisille
suunnille. Sitäpaitsi arvasin, että minulta tultaisiin kysymään oliko
kertomukseni tosi vaiko tekaistu, ja pidinkö sellaisia tapauksia
yleensä mahdollisina — ja miten näihin kysymyksiin olisin
vastannutkin, niin olisi minua pidetty joko okkultistina tai veijarina.
Senvuoksi päätin minä lopuksi kertoa yöllisen kohtaukseni tarinan
juuri sellaisena kuin se tapahtui, huolimatta siitä, että monet ihmiset
sittenkin tulevat epäilemään sen totuutta. Asian laita on nyt kerran
kaikkiaan niin, että runoilijain kertomuksia aina epäillään, vaikka
siihen on paljon vähemmän syytä, kuin useimpien muitten ihmisten
kertomusten epäilemiseen.

Uusi laulu.

"Se ei ole minun syyni, herra von Breiteneder — — — suokaa

anteeksi, sitä ei voi kukaan sanoa!" Karl Breiteneder kuuli nämä
sanat aivankuin kaukaa korviinsa kajahtavan ja tiesi kuitenkin aivan
hyvin, että se, joka ne lausui, kulki hänen vierellään — niin, tunsipa
hän sen viinilöyhkänkin, joka näitä sanoja seurasi. Mutta hän ei
vastannut. Hänen oli aivan mahdoton ruveta selittelemään; hän oli
tämän yön kauheitten tapahtumien jälkeen niin väsynyt ja järkytetty,
hän tahtoi vaan saada olla yksin ja hengittää raitista ilmaa.
Senvuoksi ei hän ollut mennyt kotiansakaan, vaan käyskenteli
mieluummin aamutuulessa autioita katuja pitkin metsäisiä kukkuloita
kohden, jotka näkyivät ylhäältä toukokuun sumun takaa. Mutta tuon
tuostakin puistatti kauhu hänen ruumistaan eikä hän tuntenut
ollenkaan sellaista aamuhetken virkeyttä, jota hän tavallisesti
yöllisten valvomisien jälkeen raittiissa aamuilmassa tunsi. Hän näki
silmänsä edessä aina vain sen kauhunkuvan, jota hän oli paennut.

Hänen vierellään kulkeva mies oli kai vasta aivan juuri hänet
saavuttanut. Mitä tahtoi hän? — — — mitä varten hän puolustautui?
— — — ja miksikä juuri hänelle? — — — Hän ei ollut suinkaan
aikonut julkisesti syyttää vanhaa Rebayta, vaikka hän kyllä hyvin
tiesi, että tämä oli suurin syypää siihen, mitä tapahtunut oli. Nyt
katseli hän häntä syrjästä päin. Miltä tuo ihminen näyttikään! Musta
lievetakki oli rutistunut ja tahrainen, yksi nappi puuttui, toiset napit
olivat reunoista rikki; eräässä napinreiässä riippui kuihtunut kukka.
Eilen illalla oli Karl nähnyt kukan vielä tuoreena. Tämä sama kukka
koristuksenaan oli kapellimestari Rebay istunut rämisevän pianon
ääressä ja säestänyt kaikkia Ladenbauer seurueen esittämiä
numeroita, niinkuin hän oli sitä tehnyt jo melkein kolmekymmentä
vuotta. Pieni ravintola oli ollut täpötäynnä kuulijoita, tuoleja ja pöytiä
oli asetettu puutarhaan saakka, sillä tänään esiintyi, niinkuin suurille,
keltaisille lehdille oli mustilla ja punaisilla kirjaimilla painettu: "Neiti
Maria Ladenbauer, 'valkoinen mustarastas' ensimäisen kerran vaikean
sairautensa jälkeen."
 Karl hengitti syvään. Päivä oli jo kokonaan valjennut, hän ja
kapellimestari eivät olleet enää yksin kadulla. Heidän takaa ja
sivukäytäviltä, sekä ylhäältä metsästäkin tuli kävelijöitä heitä
vastaan. Nyt vasta muisti Karl, että tänään oli sunnuntai. Hän oli
iloinen, kun hänen ei ollut pakko mennä kaupunkiin, vaikkakin hänen
isänsä tälläkin kertaa olisi antanut anteeksi hukkaan vietetyn
viikonpäivän, niin kuin hän jo niin usein oli tehnyt. Vanha sorvariliike
Alserkadulla pysyi vielä toistaiseksi pystyssä ilman häntäkin, ja isä
tiesi kokemuksesta, että Breitenederit olivat tähän saakka aina
oikealla ajalla päättäneet ruveta elämään säännöllisesti. Tuo juttu
Maria Ladenbauerin kanssa ei kuitenkaan ollut häntä koskaan
miellyttänyt. "Tee miten tahdot", oli hän kerran sanonut lempeästi
Kartelle, "olinhan minäkin kerran nuori — — — mutta tyttöjeni
perheitten kanssa en kuitenkaan milloinkaan ole ollut tekemisissä!
Siinä suhteessa pidin minä hyvin varani."

Jos hän olisi kuunnellut isänsä neuvoa — ajatteli Karl nyt — niin
olisi häneltä paljon ja monenlaista säästynyt. Mutta hän oli pitänyt
Mariasta niin paljon. Maria oli hyväluontoinen tyttö, piti hänestä
kovasti joutavia jaarittelematta. Ja kun Maria kulki hänen
käsikoukussaan kävelyllä, ei olisi voinut aavistaa häntä naiseksi, jolla
oli niin kirjava menneisyys takanaan. Muuten elettiin hänen
vanhempiensa kotona yhtä kunniallisesti kuin porvarillisissa
perheissä. Huoneet oli hauskasti järjestetty, hyllyllä oli kirjoja;
vanhan Ladenbauerin veli, joka oli Maistraatin virkamies tuli usein
vierailulle, ja silloin keskusteltiin hyvin vakavista asioista: politiikasta,
vaaleista ja kunnallisasioista. Sunnuntaisin pelasi Karl siellä usein
tarokkia; vanhan Ladenbauerin ja mielipuolen Jedekin kanssa,
samaisen Jedekin kanssa, joka iltasin esiintyi klownipuvussa soittaen
laseilla ja lautasien reunoilla valsseja ja marsseja; ja jos hän voitti,
maksettiin hänelle raha ilman muuta, mikä ei suinkaan tapahtunut
säännöllisesti hänen kahvilassaan. Akkunasyvennyksessä, jossa
riippui sveitsiläisiä maisema-lasikuvia, istui kalpea ja pitkä rouva
Jedek, joka iltasin esitti näytännössä pitkäveteisiä runoja, jutteli
Marian kanssa ja nyökäytti päätään herkeämättä. Mutta Maria katsoi
vastapäätä istuvaa Karlea, tervehti häntä ilakoiden kädellään tai
istuutui hänen viereensä ja katseli hänen kortteihinsa. Marian veli
palveli eräässä suuressa liikkeessä, ja kun Karl tarjosi hänelle
sikaarin, tarjosi hän heti vuorostaan. Hän toi sisarelleen, josta hän
kovasti piti, joskus kaupungin sokurileipurista makeisia. Ja kun hän
poistui sanoi hän silmät puoleksi sulettuina: "Ikävä, että olen
lupautunut muualle — — —"

Karl oli tietysti mieluimmin yksin Marian kanssa. Ja hän muisti

erään aamun, jolloin hän oli kävellyt Marian kanssa tätä samaa tietä,
jota hän nyt kulki, hiljaisesti suhisevaa metsää kohden, joka tuolta
ylhäältä kukkulalta alkoi. He olivat molemmat olleet silloin väsyneitä,
sillä he tulivat suoraan kahvilasta, missä he olivat istuneet koko
kansanlauluseurueen kanssa varhaiseen aamuun saakka; sitten
paneutuivat he lepäämään erään niityn reunassa olevan pyökin alle
ja nukahtivat. He heräsivät vasta myöhään kesäpäivän kuumaan
hiljaisuuteen, jatkoivat matkaansa yhä syvemmälle metsään,
juttelivat ja nauroivat koko päivän, tietämättä miksi, ja vasta
myöhään illalla toi hän tytön jälleen kaupunkiin iltaesitykseen — —
—. Tällaisia ihania muistoja oli paljon, ja molemmat elivät he hyvin
tyytyväisinä ajattelematta ollenkaan tulevaisuutta. Mutta talven
alussa sairastui Maria äkkiä. Tohtori oli ankarasti kieltänyt kaikki
vieraskäynnit, sillä tyttö sairasti aivotulehdusta tai jotain muuta
senkaltaista ja jokaista mielenliikutusta täytyi välttää. Karl kävi aluksi
joka päivä Ladenbauerien luona tiedustelemassa sairaan tilaa; mutta
myöhemmin, kun sairaus yhä pitkittyi, kävi hän vain joka toinen tai
joka kolmas päivä. Kerran sanoi rouva Ladenbauer hänelle ovessa:
"Tänään uskallatte jo tulla sisään, herra von Breiteneder. Mutta
muistakaa, ettette paljasta itseänne." — "Millä tavoin minä
paljastaisin itseni?" kysyi Karl, "mitä on tapahtunut?" — "Niin, hänen
silmänsä ovat ihan parantumattomat." — "Kuinka niin?" — "Hän ei
näe enää mitään — — —, sairaus sen teki. Mutta hän ei itse vielä
tiedä, että se on parantumatonta — — — Varokaa tekin, ettei hän
mitään huomaa." Silloin änkytti Karl vain pari sanaa ja lähti pois.
Hänet valtasi äkkiä pelko nähdä Maria jälleen. Hänestä tuntui, ettei
hän ollut mitään niin paljon rakastanut kuin Marian silmiä, jotka aina
olivat olleet niin kirkkaat, ja hymyilevät. Hän aikoi tulla huomenna
uudestaan. Mutta hän ei tullut, ei seuraavana eikä vielä sitä
seuraavanakaan päivänä. Ja yhä uudelleen siirsi hän käyntinsä
myöhemmäksi. Hän tahtoi tavata Marian vasta sitten, näin ajatteli
hän, kun Maria itse oli tottunut alistumaan kohtaloonsa. Ja sitten
sattui niin, että hänen täytyi lähteä eräälle liikematkalle, jonne isä jo
kauvan oli kärttänyt. Hän joutui kauvas, hän kävi Berlinissä,
Dresdenissä, Kölnissä, Leipzigissä ja Pragissa. Kerran kirjoitti hän
vanhalle rouva Ladenbauerille kortin, jossa hän mainitsi heti kotiin
palattuaan tulevansa tervehtimään, ja lähetti Marialle paljon
terveisiä. — Keväällä palasi hän kotia; mutta Ladenbauerin luona ei
hän käynyt. Ei tullut lähdetyksi — — — Hän ajatteli Mariata päivä
päivältä luonnollisesti yhä vähemmän ja tahtoi hänet kokonaan
unohtaa. Eihän hän ollut ensimäinen eikä liioin ainoakaan Marian
ystävä. Hän ei myös kuullut Mariasta mitään ja rauhottui yhä
enemmän ja jostakin syystä kuvitteli hän usein, että Maria asui
maalla sukulaistensa luona, joista hän oli kuullut Marian usein
puhuvan.

Mutta eilen illalla vei sattuma hänet — hän tahtoi käydä lähellä
asuvien tuttaviensa luona — sen ravintolan ohitse, jossa
Ladenbauerin seurue tavallisesti antoi näytäntönsä. Hän aikoi jo
ajatuksissaan sivuuttaa ravintolan, mutta silloin näki hän keltaisen
ilmoituslehden ja huomasi, missä hän oli, ja sydämessään tunsi hän
pistoksen, ennenkuin hän oli sanaakaan lukenut. Mutta luettuaan
mustilla ja punaisilla kirjaimilla painetun ilmoituksen: "Maria
Ladenbauerin, niin kutsutun 'valkoisen mustanrastaan' ensimäinen
esiintyminen parantumisensa jälkeen", jäi hän kuin halpautuneena
seisomaan. Ja samassa hetkessä seisoi Rebay hänen vieressään
aivan kuin maasta nousseena; valkoinen pörröpää paljaana, kulunut
silinterihattu kädessä ja napinlävessä tuore kukka. Hän tervehti
Karlea: "Herra Breiteneder — ihan omassa persoonassaan!
Kunnioitatteko meitä tänään läsnäolollanne jälleen? Maria neiti
ilostuu varmaan ihan ikihyväksi, kun hän kuulee, että entiset
ystävänsä häntä vielä muistavat. Lapsi parka? Meillä on ollut hänen
kanssaan kovat ajat, herra von Breiteneder; mutta nyt se on ohi."
Rebay jutteli vielä yhtä ja toista ja Karl seisoi paikallaan, vaikka hän
olisikin mieluimmin tahtonut olla kaukana poissa. Mutta äkkiä heräsi
hänessä uusi toivo, ja hän kysyi Rebaylta, oliko Maria ihan sokea —
eikö hän nähnyt vähääkään. "Nähnyt vähääkään?" vastasi toinen.
"Mutta mitä te ajattelette herra von Breiteneder!" — — — iloisesti.
"Koko maailma on pikimusta hänelle — — — Mutta Te tulette siitä
vakuutetuksi, herra von Breiteneder, että kaikella on hyvätkin
puolensa, jos niin saa sanoa — ja ihana ääni sillä tytöllä on,
kauniimpi kuin koskaan ennen! — — — No niin, Te saatte itse sen
nähdä, herra von Breiteneder. — Ja hyvä hän on — ihmeen hyvä!
Vielä paljoa ystävällisempi, kuin tätä ennen. No, no, Tehän tunnette
hänet — ha, ha! — Niin, niin, tänään tulee sinne useampia sellaisia,
jotka tuntevat hänet — luonnollisesti ei niin hyvin kuin Te, herra von
Breiteneder, sillä nyt eivät sellaiset asiat luonnollisesti enää tule
kysymykseen. Mutta ehkenpä se aika vielä taas tulee! Minä tunsin
erään, joka oli sokea ja sai kaksoset — ha, ha! — Mutta
katsokaapas, kuka tuolla on." sanoi hän äkkiä, sillä he seisoivat
kassaluukun edessä, jonka takana istui rouva Ladenbauer. Hän oli
pöhöttynyt ja kalpea ja katsoi Karlea sanaakaan sanomatta. Hän
antoi Karlelle lipun, jonka Karl maksoi oikeastaan tietämättä, mitä
hän teki. Mutta äkkiä sanoi hän: "Elkää sanoko Marialle mitään,
Jumalan tähden, elkää sanoko rouva Ladenbauer — — — ei
sanaakaan Marialle siitä, että minä olen täällä! — — — Herra Rebay,
ei sanaakaan Marialle!"

"Hyvä on," sanoi rouva Ladenbauer ja kääntyi toisten lippuja

kysyvien puoleen.

"Minä en sano sanaakaan", sanoi Rebay. "Mutta jälestäpäin tulee

yllätys! Tulettehan mukaan? Suuri juhla — hohoo! Sulkeudun
suosioonne, herra von Breiteneder." Ja hän oli kadonnut. Karl kulki
täpötäyden salin läpi ja istuutui salin yhteydessä olevan puutarhan
perälle erään pöydän ääreen, jossa jo ennestään istui kaksi vanhaa
ihmistä, mies ja vaimo. Nämä eivät puhuneet sanaakaan keskenään,
katsoivat vaan ääneti uutta vierasta, ja nyökkäsivät surumielisinä
toisilleen. Karl istui ja odotti. Esitys alkoi, ja Karl kuuli ikivanhat asiat
uudelleen. Hänestä näytti kaikki vaan niin kummallisen
muuttuneelta, sillä hän ei ollut koskaan ennen istunut näin etäällä
näyttämöltä. Kapellimestari Rebay soitti ensin niin kutsutun
uvertyyrin, josta Karl kuuli vain muutamia kovia akordeja, sitten
esiintyi ensimäisenä unkaritar Ilka kirkkaanpunaisessa puvussa,
kannukset jalassa ja lauloi unkarilaisia lauluja ja tanssi czardasta.
Tämän jälkeen seurasi ilveilijä Wiegel-Wagelin humoristinen esitys;
hän oli puettu viheriään hännystakkiin ja ilmoitti juuri saapuneensa
Afrikasta sekä kertoi kaikenlaisia hullunkurisia seikkailujuttuja, jotka
loppuivat hänen naimiskauppaansa erään vanhan lesken kanssa.
Sitten lauloivat rouva ja herra Ladenbauer duetin; molemmat olivat
tyroolilaispuvuissa. Heidän jälkeensä esiintyi likaiseen, valkoiseen
klovnipukuun puettu hupsu pikku Jedek näytellen ensin
taikatemppujaan, sitten harhaili hän silmät suurina väkijoukon
keskellä, aivankuin hän olisi jotain etsinyt; sitten asetti hän lautasia
eteensä riviin, ja takoi niitä puupalikalla soittaen marssia, järjesteli
laseja ryhmiin ja soitti kosteilla sormillaan niitten reunoja kosketellen
alakuloista valssin säveltä. Sitä tehdessään katsoi hän ylös kattoon ja
hymyili autuaallisesti. Hän poistui ja Rebay hakkasi uudelleen
soittokoneesta juhlallisia säveliä. Ympäri salia ja puutarhaa kuului
kuisketta, ihmiset kurottivat päitään, ja äkkiä seisoi Maria
näyttämöllä. Hänen isänsä, joka oli taluttanut hänet sinne, oli heti
jälleen hävinnyt; ja Maria seisoi siinä yksin. Karl näki hänet siinä
seisomassa — sammuneet silmät suloisissa, kalpeissa kasvoissa; hän
näki aivan selvään, kuinka hän ensin liikutti vain huuliaan ja hieman
hymyili. Karl oli huomaamattaan noussut ylös ja nojautui viheriää
lyhtyä vasten ja oli vähällä parahtaa säälistä ja tuskasta. — Nyt alkoi
Maria laulaa. Aivan oudolla äänellä, hiljaa, paljon hiljempaa kuin
ennen. Se oli sama laulu, jota Maria oli aina laulanut ja jonka Karl oli
kuullut vähintään viisikymmentä kertaa, mutta ääni oli hänestä
sittenkin kummallisen vieras, ja vasta kun Maria lauloi loppukerron
"Mua kutsuvat he valkoiseksi mustaksi rastaaksi kylässä ja
kotonakin", luuli hän tuntevansa äänen samaksi jälleen. Maria lauloi
kaikki kolme värssyä ja Rebay säesti häntä tapansa mukaan katsoen
usein ankarasti Mariaan. Kun laulu oli lopussa, puhkesi kova ja
myrskyävä suosionosotus. Maria hymyili ja kumarsi. Äiti nousi kolme
rappusta näyttämölle, Maria tavoitteli kädellään ilmaa, aivankuin
äitinsä käsiä etsiäkseen, mutta suosionosotus oli niin voimakas, että
hänen täytyi heti laulaa toinen laulunsa, jonka Karl myöskin oli jo
melkein viisikymmentä kertaa kuullut. Se alkoi näin: "Tänään menen
minä kultani kera maalle — — —", ja Maria heitti päätään niin
tyytyväisenä taaksepäin, keinutti ruumistaan kevyesti edes takaisin,
aivankuin hän todellakin vielä voisi kultansa kanssa mennä maalle,
voisi nähdä sinisen taivaan ja viheriät niityt ja voisi tanssia ulkona
vapaassa luonnossa, niin kuin hän laulussa lauloi. Ja sitten lauloi hän
kolmannen, uuden laulunsa. —

"Täällä olisi pieni puutarha", sanoi herra Rebay, ja Karl vavahti. Oli
kirkas päivänpaiste; katu loisti auringonpaisteessa ja ympärillä oli
valoisaa ja vilkasta. "Sinne voisi pistäytyä levähtämään", jatkoi
Rebay "ja juoda lasin viiniä; minulla onkin jo kova jano — tänään
tulee kuuma päivä."

"Kuuma päiväkö?" sanoi joku heidän takanaan. Breiteneder

kääntyi katsomaan — — Kuinka, oliko tuokin nyt juossut hänen
jälkeensä? — — — Mitä tuo sitten hänestä tahtoi? — — — Se oli
hupsu Jedek; häntä ei oltu koskaan kutsuttu muulla nimellä, ja aivan
varmaa olikin, että hän pian todellakin oli tuleva kokonaan hulluksi.
Pari päivää sitten oli hän aikonut tappaa pitkän, kalpean vaimonsa,
ja oli aivan ihmeellistä, että hänen annettiin käydä vapaana. Nyt hiipi
hän kääpiömäisen pienenä Karlen rinnalla; kellertävistä kasvoista
katselivat mulkoillen omituisen hassunkuriset silmät, päässä oli
hänellä yleisesti tunnettu harmaa, pehmeä hattu, jossa oli kulunut
sulka, ja kädessä ohut kävelykeppi. Ja nyt oli hän noitten toisten
edellä pujahtanut pieneen ravintolapuistoon, istuutunut puupenkille,
joka nojasi matalaa talonseinää vasten, ja löi kävelykepillään kiivaasti
viheriäksi maalattua pöytää huutaen tarjoilijaa. Molemmat toiset
seurasivat häntä. Valkoinen tie, jota reunusti viheriä puuaita jatkui
pienten, surkeitten huvilain ohitse ylös vuorelle, ja katosi metsään.

Tarjoilija toi viiniä. Rebay asetti silinterinsä pöydälle, silitteli

valkoista tukkaansa, hieroi molemmilla käsillään tapansa mukaan
sileitä poskiansa, työnsi Jedekin lasin syrjään ja kumartui pöydän yli
Karlen puoleen. "Enhän minä mikään pölkkypää ole, herra von
Breiteneder! Tiedänhän minä, mitä minä teen! — — — Miksikä minä
olisin siihen syypää? — — — Tiedättekö Te, kenelle minä
nuoruudessani kirjoittelin kupletteja? — — — Kuuluisalle Matras'ille!
Se ei ole mikään pikku asia! Ne herättivät huomiota! Sanat ja
musiikki minun! Ja useita niistä sijoitettiin toisiin kappaleihin!"

"Antakaa lasin olla", sanoi Jedek hiljaa hihittäen.

"Hyvä herra von Breiteneder", jatkoi Rebay työntäen lasin

uudestaan luotaan. "Tehän tunnette minut ja tiedätte, että minä olen
kunniallinen mies! Eikä minun kupleteissani ole milloinkaan ollut
mitään sopimatonta eikä rivoa! — — — Se kupletti, jonka vuoksi
vanha Ladenbauer aikoinaan sai sakkoa, oli toisen tekemä! — — Ja
tänään olen minä kuudenkymmenenkahdeksan vuoden vanha, herra
von Breiteneder — se merkitsee jo jotain! Tiedättekö Te, kuinka
kauvan olen jo kuulunut Ladenbauerien seurueeseen? — — — Silloin
eli vielä Eduard Ladenbauer, joka oli perustanut seurueen. Ja Marian
tunnen minä syntymästään saakka. Kaksikymmentäyhdeksän vuotta
olen minä ollut Ladenbauerin seurueessa — ensi maaliskuussa on
minun riemujuhlani — — — Enkä minä ole varastanut laulujeni
säveleitä — kaikki olen itse tehnyt, ihan kaikki! Ja tiedättekö, miten
monta niistä on aikoinaan esitetty? — — — Kahdeksantoista! Eikö
totta, Jedek? — — —"

Jedek nauroi yhä edelleen äänetöntä nauruaan, silmät selällään.

Hän oli kerännyt eteensä kaikki kolme lasia ja alkoi nyt sormellaan
kevyesti silitellä lasien reunoja. Hänen soittonsa oli hienoa, hieman
liikuttavaa, aivankuin kaukainen oboen tai klarinetin ääni.
Breiteneder oli aina kovasti ihaillut tätä hänen taituruuttaan, mutta
tällä hetkellä tuntui se hänestä sietämättömältä. Muissa pöydissä
istuvat ihmiset kuuntelivat soittoa; muutamat nyökkäsivät päätään
tyytyväisinä, eräs paksu herra paukutti käsiään. Äkkiä työnsi Jedek
taasen kaikki lasit luotansa, pani kätensä ristiin rinnalle ja tuijotti
valkoiselle tielle päin, joka yhä enemmän täyttyi metsään päin
kävelevistä ihmisistä. Karlen silmät säkenöivät ja hänestä tuntui, että
kaikki ihmiset tanssivat ja pyörivät hämähäkinverkon takana. Hän
hieroi ohimoitaan ja silmäluomiaan, hän tahtoi päästä jälleen
tajuihinsa. Eihän hän voinut sille mitään! Se oli kauhea onnettomuus
— mutta eihän se ollut hänen syynsä! Ja äkkiä hän nousi ylös, sillä
kun hän ajatteli loppua oli hänen rintansa pakahtua. "Mennään
pois", sanoi hän.

"Mennään vaan, raitis ilma on pääasia", vastasi Rebay.

Jedek oli äkkiä suuttunut, kukaan ei tiennyt, miksi. Hän asettui

erään pöydän luo, jonka ääressä istui rauhallinen pari, hutki
kävelykepillään ympärinsä ja huusi kovalla äänellä: "Hitto ruvetkoon
lasinsoittajaksi, tuhat tulimmaista!" Molemmat rauhalliset ihmiset
tulivat hämilleen ja koettivat tyynnyttää häntä; toiset nauroivat ja
luulivat häntä juopuneeksi.

Breiteneder ja Rebay olivat jo saapuneet valkoiselle, kadulle, ja

Jedek, jälleen rauhoittuneena, hyppelehti heidän jälessään. Hän otti
harmaan hattunsa päästään ja ripusti sen kävelykeppinsä nenään ja
piteli keppiä hattuineen olkapäillä niinkuin pyssyä, tehden toisella
kädellään sillä aikaa suuria tervehdysliikkeitä taivasta kohden.

"Teidän ei pidä luulla, että niinä koetan itseäni puolustella," sanoi

Rebay kalisevin hampain. "Ee-eh, minulla ei ole siihen mitään syytä!
Ei ollenkaan! Minulla oli mitä paras tarkoitus, ja jokaisen täytyy se
myöntää. Itsehän minä harjoittelin tuon laulun hänen kanssaan! —
— — Vieläpä silloin kun hän istui huoneessaan silmät siteissä. Ja
tiedättekö, miten tuo aihe johtui minulle mieleen? Tässä on nyt
tapahtunut onnettomuus, sanoin itsekseni, mutta kaikki ei ole vielä
hukassa. Hänellä on vielä kaunis äänensä ja kauniit kasvonsa — —
— Minä sanoin sen äidillekin, joka oli ihan epätoivoissaan. Rouva
Ladenbauer, sanoin minä hänelle, vielä ei ole mitään kadotettu —
odottakaahan vaan! Nykyään, kun on noita sokeain laitoksiakin,
jossa sokeat voivat vähitellen oppia lukemaan ja kirjoittamaankin —
— — Minä tunsin erään — nuoren miehen, joka kahdenkymmenen
vuoden vanhana tuli sokeaksi. Hän uneksi joka yö mitä ihanimmista
ilotulituksista ja kaikenlaisista mahdollisista valaistuksista — — —"

Breiteneder naurahti. "Puhutteko Te tosissanne?" kysyi hän

Rebaylta.

"Kuinkas muuten!" vastasi Rebay raa'asti, "mitä Te oikeastaan

tahdotte? Pitäisikö minun tappaa itseni? — — — Ja miksi? — Hyvä
Jumala, minä olen ollut kyllin onneton tässä maailmassa! — Tai
luuletteko Te, herra von Breiteneder, että se on mitään elämää, kun
nuorena on kirjoittanut kerran näytelmäkappaleen, niinkuin minäkin
ja sitten kuudenkymmenenkahdeksan vuoden vanhana on lopuksi
niin pitkällä, että parista kurjasta kreuzerista täytyy huonolla
pianorähjällä säestää käheä-äänisiä retkaleita ja kirjoittaa heille
kupletteja — — — Tiedättekö Te, paljonko minulle yhdestä kupletista
maksetaan? — — — Teitä se varmaankin ihmetyttäisi, herra von
Breiteneder!"

"Mutta niitähän lauletaan ravintoloissa", sanoi Jedek, joka käveli

nyt heidän rinnallaan aivan totisena ja siivona, melkeinpä hienon
miehen tavoin.
 "Mitä Te oikeastaan minusta tahdotte?" sanoi Breiteneder. Hänestä
tuntui äkkiä, että nuo molemmat vainosivat häntä, mutta hän ei
tiennyt, minkä tähden. Mitä tekemistä hänellä oli noitten ihmisten
kanssa? — — — Mutta Rebay vaan jatkoi: "Minä tahdoin hankkia
tytölle toimeentulon! — — — Turvata hänen tulevaisuutensa,
näettekös! — — — Ja juuri tuolla uudella laululla! — — Juuri sillä! —
— — Eikö se sitten muka ole kaunis? — — Eikö se ole liikuttava? —
— —"

Pikku Jedek tarttui äkkiä Breitenederin takin hihaan ja pidätti

häntä, nosti vasemman kätensä etusormen ylös, huomaavaisuutta
pyytäen, pani huulensa suppuun ja vihelsi uuden laulun säveltä, jota
Maria Ladenbauer, jota myös kutsuttiin valkoiseksi "mustaksi
rastaaksi", edellisenä yönä oli laulanut. Hän vihelsi sitä kerrassaan
mestarillisesti; sillä viheltäminen kuului myös hänen taitoihinsa.

"Ei se ollut sävelen vika", sanoi Breiteneder.

"Kuinka?" huusi Rebay. — He kulkivat kaikki kiivaasti, melkein

puolijuoksua, huolimatta tien jyrkästä noususta. "Kuinka herra von
Breiteneder? — — — Sanatko siinä siis olivat sopimattomat? — — —
Hyvä Jumala, eihän niissä sanoissa ole mitään muuta, kuin sellaista,
minkä Maria itse tiesi? — — — Ja kun minä hänen omassa
huoneessaan harjoittelin sitä hänen kanssaan, niin ei hän kertaakaan
itkenyt. Hän sanoi vaan: Se on surullinen laulu, herra Rebay, mutta
kaunis se on! —" "Kaunis se on", sanoi hän — — — "Niin, se on
tosiaankin surullinen laulu, herra von Breiteneder — mutta onhan
hänen kohtalonsakin surullinen. Olisinko minä silloin voinut kirjoittaa
mitään iloista laulua hänelle? — — —"

Tie häipyi metsään. Aurinko kimalteli oksien lomista; pensaista

kuului naurua ja huutoja. He kulkivat kaikki kolme vierekkäin, niin
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