BioChemLab Exp 1,2,3 2/11/15 11:33 PM

Experiment 1: Isolation and hydrolysis of Casein from milk (non-fat)

Protein
o Polymer with monomer unit called amino acids
o By peptide bonds
o Easy to separate from other molecules but difficult from each
other
o Separated by diff. in size,charge, ability to bind
o Types:
§ Fibrous – form long and strong repeated sets of aa
residues
§ Globular – fold back on themselves into compact units
approach spheroidal shapes

Milk
o Complex bio fluid w/ high amt of proteins,lipids,minerals
o Source of Ca and K; deficient in Fe and ascorbic acid
o 87.1% water; 3.4% protein; 3.9% fats; 4.9%
carbohydrates; 0.7 % minerals

Carbohyd
Wate rates   Lipid
r   Milk   s  
composi
Minera tion   Prote
ls   Vitami ins  
ns  
o Proteins in Milk: Globular and complete proteins
§ Lactoglobulin – immunological
§ Lactoalbumins – soluble in H20 & salt soln; denatured
by heat
§ CASEIN- main protein (80%)
CASEIN
o Phosphoproteins
o No disulfide bridges
o Isoelectric point = pH 4.6
o Hydrophobic: insoluble in water, ROH & ROR ; soluble in
dilute alkalis/ salt solutions
o Causes white color of milk with lipids
o Exists as calcium salt & calcium caseinate
o Contais essential aa (complete protein)
 o Heterogenous mixture:
§ α-Caseins are the major casein proteins. Its containing
8-10 phosphate groups
§ β- casein contains about 5 phosphate residues
§ κ-caseins are glycoproteins, and they have only 2
phosphate group
§ MICELLE
ú Solubilized unit
ú Colloidal suspension
Hydrolysis
o involves breaking the peptide bonds through the addition of
water (H20)
o requires high temperatures and either strong acid or strong
base
o types:
§ Acid Hydrolysis
§ Base/Alkaline Hydrolysis
§ Enzymatic Hydrolysis

Neutralization
o acid + base -> H2O + salt
o H20 (H+ ions and OH- ions combination)

Objectives of exp:
o isolate casein by isoelectric precipitation
o subject to acid & alkaline hydrolysis
o neutralize the hydrolyzate

Methodology
Results& Discussion

Isolation of casein
§ used non-fat milk to lessen contents to be separated
§ heat at 55 degrees C to prevent unnecessary
denaturing of protein content
§ added acetic acid until pH = 4.6 -> isolectric point for
precipitation
§ Principle:
ú casein has negative charge at typical pH of milk
which makes it soluble in the solution
ú by adding Ac acid -> neutral -> insoluble ->
precipitate

Acid and Base Hydrolization & Neutralization

§ bond cleavage of chem bonds simultaneous with

addition of H2O
§ break peptide (amide) bonds in intact proteins
§ hydrolyzed by STRONG ACID/BASE

§ Acid hydrolysis
 ú Protic acid -> catalyze the cleavage of bond with
addition of water
ú Strong acids: H2SO4, HCl, HNO3, HClO4
ú Acid as catalysts
ú Without racemization of C-configuration
ú Trp (W) is destroyed into humin (black)
ú Thr (T) and Ser(S) ara destroyed
ú Asn (N) → Asp (D), Gln (Q) → Glu (E)
ú In the experiment used H2SO4 rather than HCl
because ______________

§ Base Hydrolysis
ú Destroys more amino acids
ú STRONG BASES: Ba(OH) 2, NaOH, KOH
ú Trp (W) is not destroyed
ú The base, Ba(OH) 2, also acts as a catalyst
beneficial in complete removal of sulfate ions
because the BaSO4 formed is insoluble in water
and precipitates out.

ú Promotes racemization
ú Thr (T) and Cys (C) are lost
 ú Arg (R) is destroyed and converted to urea &
ornithine = yellow-orange color

§ Autoclave
ú Heat catalyzed reaction
ú Frees amino acid in high pressure as the acid/
base speeds up the hydrolysis
ú To hasten the hydrolysis of reaction
§ Dilute hydrolyzate
ú Reduce the H2SO4 concentration to a value of not
more than 6N
§ Neutralization
ú Reagents used:
• Acid hydrolysis -> Ba(OH) 2
• Base hydrolysis -> H2SO4
• Ba(OH) 2
• → beneficial in complete removal of sulfate
ions because the BaSO4 formed is insoluble
in water and precipitates out.
 2/11/15 11:33 PM

EXPERIMENT 2 : Color Reaction of Intact Protein and Hydrolyzate

Objectives
o to analyze the functional groups from isolated casein from
low-fat milk and acid/basic hydrolyzate used in color reactions
o to explain the principles behind each test

Tests:
o Buiret test
§ general test for peptide bonds
§ reagents:
ú 0.01 M CuSO4 solution - gives off copper(II) ions
which reacts with amide N atoms.
ú 2.5 M dilute NaOH - form an alkaline solution
§ positive result: PURPLE SOLUTION
§ Principle: Complexation of Cu+ with Amide N atoms
§ Results:
ú Intact Protein- (+)
ú Acid and Base – (-) since bond is cleaved; copper
makes it blue meaning NR

o Sakaguchi test
§ this color reaction test specifically detects the presence
of arginine (contains guanidino group)
§ REAGENTS:
ú a-naphthol solution - induces condensation
reaction.
ú NaOH - make amino acid zwitterionic and reach
its isoelectric point
ú NaOBr (and urea to stabilize color and destroy
excess OBr- anions)- oxidizing agent.
§ Positive result: red to red-orange color
§ PRINCIPLE: base-catalyzed condensation of alpha-
naphthol with the guanidino group of arginine
 § Results:
ú Intact Protein & Acid – (+)
ú Base – (-) since Arg (R) is destroyed

o Ninhydrin test
§ general test for compounds with free a-amino group
§ free amino acid group and free carboxylic acid group
§ REAGENT:
ú Ninhydrin solution- reacts with amino grp of
guanidine
ú Water – hydrolysis
§ Positive result: blue-to-purple color (Ruhemann’s
purple)
§ Principle: oxidative deamination and decarboxylation
of amino group, reduction of ninhydrin
§ Results:
ú Intact Protein, Acid, Base – (+)
 o Xanthoproteic test
§ general test for aromatic amino acids
§ REAGENTS:
ú conc HNO3
ú conc. NaOH
• neutralize excess acid
• confirming xanthoproteic acid
§ Positive Result: Yellow or orange solution
§ Principle: Nitration of Aromatic ring by aromatic
electrophilic substitution
§ Intact Protein, Acid, Base – (+)

Intact Protein: 4 HNO3 → 2 H2O + 4 NO2 + O2.

o Hopkins-Cole test
§ detects the presence of indole group in tryptophan

§ REAGENTS:
ú Magnesium
ú oxalic acid
ú Hopkins-cole reagent- as glyoxylic acid
 
ú conc. H2SO4 - The protein solution is hydrolyzed
at interphase
ú Positive Reaction: pink to violet interphase
ú Principle: Condensation of indole group with
glyoxylic acid and H2SO4
ú Results:
• Intact Protein & Acid interphase (+)
• Base – (-) – Trp (W) destroyed
 O H
O H
O
O
-­‐
N H 3 Mg O O

O O H 3 N O O H
-­‐
O + O H H
+
H H
N
O N
H N
H
O

-­‐ N H 3
O

OVERVIEW:

Test% Intact%Protein% Base%Hydrolyzate% Acid%Hydrolyzate%

Biuret%Test% +" #" #"
Sakaguchi% +" #" +"
Ninhydrin% +"" +" +"
Xanthoproteic% +" +" +"
Hopkins=Cole%% +" +" #"
"
2/11/15 11:33 PM