Name: Shalinie Arjune

 
ID Number: 816008336
 
Date: 28th April 2020.
 
Course: BIOC 2169

Title: Partial Purification and characterization of Yeast Invertase (Enzyme Kinetics and SDS-
PAGE) 
Aim:
·  To determine The effect of enzyme concentration on initial velocity for Yeast
Invertase
·  To investigate the effect of incubation time on product formation for yeast invertase
·  To evaluate the effect of substrate concentration on enzyme activity in the presence
and absence of the inhibitor urea for yeast invertase.
·  To categorise the inhibitor Urea on the basis of kinetic data (Lineweaver-Burk plot)
·  To determine the kinetic parameters for yeast invertase; Vmax and Km
·  To conduct a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on the enzyme
fractions obtained from each purification step.
·  To observe the purity differences of each collected enzyme fraction, using SDS-
PAGE
 

Introduction:

       Enzymes are biological catalysts, that catalyzes or speed up the chemical reactions in a
organism, ideally enzymes lower the activation energy of a reaction. In biochemistry, enzyme
kinetics is the study of how enzymes affects the rate of a reaction. This is represented in a
graph which is  known as the Michaelis-Menten graph, which is shown below in Fig.1,
(KhanAcademy.com, 2020). The energy kinetic parameters on these graphs, are observed
when a plot of the reaction rate versus the substrate concentration reveals V , which is the
max

maximum rate of the reaction when there is an excess of substrate or substrate saturation,
where as   K is the substrate concentration at which the rate of reaction is half that of the V , 
m, max

it is also referred to as the Michaelis-Menten constant (PhysiologyWeb, Dec 8th 2014). It is

stated that the K is inversely proportional to the affinity of the enzyme/transport to the
m

substrate, as a result, a low K is associated to a high affinity of enzyme to substrate, as a low

m

concentration or small amount of substrate is needed for substrate saturation. 

Fig..1 The Michaelis-Menten Graph Plot, as well as the Michaelis Menten equation, 
taken from Koning, Ross E. 1994. Enzyme Kinetics. Plant Physiology Information
Website. http://plantphys.info/plant_physiology/enzymekinetics.shtml. (4-24-2020).

          Factors which primarily affect enzyme kinetics are the concentrations of the enzymes
and/or substrate, temperature, pH, activators, referred to as coenzymes and cofactors,
inhibitors, time, UV light and radiations as well as oxidizing agents (Dr. Rohini C Sane, Dec
2nd 2016). The concentration of enzymes substrate and enzymes have a major role to play as
it is linked to the velocity, therefore the rate at which products are produced is determined by
the substrate and enzyme concentrations, when a plateau is achieved, this is in the presence of
substrate saturation. Temperature affects enzyme kinetics as there temperature can either
allow enzymes to work efficiently, this is known as optimum temperature or completely
damage or denature an enzyme, i.e. the enzyme is no longer functional as the optimum
temperature is exceeded, this may be due to the enzyme losing its structure as bonds may
break due to the high temperatures which the protein cannot withstand. 
    Enzyme inhibition is a major control of biological systems (Luca Mazzei et al, 2016), it
refers to the ways in which enzyme activity are regulated experimentally or naturally, (N.V.
Bhagavan et al, 2011).  There are three main types of inhibitors that are known in enzyme
kinetics, these are competitive inhibition, non-competitive inhibition and uncompetitive
inhibition. 
Competitive inhibitors are considered to be reversible inhibitors, where these inhibitors bind
to the active sites of the enzyme preventing the substrate from binding. The competitive
inhibitors have to compete with the substrate for a place to bind with on the enzyme, as a
result this increases the K , this increase would cause the graph plot to be shifted to the right
m

as seen in Fig 5,   however the V stays the same or is unaffected. Also, on the Lineweaver-
max  
Burk graph, the plot would be steeper compared to that of no inhibitors, this is observed in
Fig 3 below.

Fig 3, Graph showing competitive inhibition on a Lineweaver-Burk plot taken from

Koning, Ross E. 1994. Enzyme Kinetics. Plant Physiology Information Website.
http://plantphys.info/plant_physiology/enzymekinetics.shtml. (4-24-2020).

Non-competitive inhibition, the inhibitors bind to another site on the enzyme, which would
result in a physical or structural change of the substrate binding site, as a result there is a
decrease in V , which is represented by a lower maximum on the Michaelis-Menten plot, K
max m

is unaffected or unchanged. Also when the plot is done on a Linewearver-Bruk graph there is
a higher y-intercept seen as compared to a plot with no inhibitor, which is shown below in
Fig 4. 
Fig 4, Graph showing non-competitive inhibition on a Lineweaver-Burk plot taken from
Koning, Ross E. 1994. Enzyme Kinetics. Plant Physiology Information Website.
http://plantphys.info/plant_physiology/enzymekinetics.shtml. (4-24-2020).

      Lastly uncompetitive inhibition is different from both competitive and non-competitive,

as in this type of inhibition the V  and K are both reduced as the inhibitor itself, binds to the
max m

enzyme-substrate or ES complex, as a result forms an enzyme-substrate-inhibitor complex or

ES-I complex. The ES-I. The ES-I cannot release the product as the inhibitor is still bound,
this is the reason for the decreased V , however the K decreased is explained by the
max m 

presence of the inhibitor on the ES complex, reduces the concentration of the ES complex
present, based on Le Chatelier;s principle, there is a shift to form additional ES complexes,
this is due to there being less free enzymes and more ES and ES-I complexes. Due to the
decrease in free enzymes, there is a greater affinity for an enzyme to be paired with its
substrate, as a result decreases the  K , but increases an affinity of enzyme to substrate.
m

        Enzyme kinetics were investigated in Enzyme Kinetics Using Fraction 4 as experiment

to determine the effect of enzyme concentration on the initial velocity and the effect of
incubation time on product formation, were conducted and studied for invertase to determine
the optimum standards for the aforementioned variables, to determine the effect of the
substrate concentration on the rate of hydrolysis of sucrose in the absence and  presence of an
inhibitor. The enzyme kinetics using Fraction 4 and Acrylamide Electrophoresis, however
was carried out to determine the degree of inhibiton on the invertase-catalyzed hydrolysis of
sucrose and to also determine the effect of an increase in substrate concentration on the rate
of enzyme activity. 

Electrophoresis is a process by which particles are charged in order for them to move to
opposite charged electrodes, electrophoresis is commonly carried out in gels as they as
a molecular sieve that would enhance separation of the molecule. Smaller molecules
readily move through the gel when they are smaller than the pores, compared to larger
molecules which are almost immobile and get stuck, however medium sized molecules
or intermediate sized molecules  Polyacrylamide gels are the preferred materials to act
as the supporting media in many separations as they are chemically inert and are readily
formed by the polymerization of acrylamide. In SDS-PAGE, 

Coomassie Blue is commonly used in the lab for protein quantification as it works by
fixing proteins, it binds to the proteins via Van der Waals interactions, through ionic
interactions between dye sulfonic acid group and positive protein amine groups
(Protocols Online, 14th July 2012). The common stains are either the G-250 (colloidal)
or R-250 form of the dye. Colloidal coomassie stains are formed to effectively stain
proteins in approximately 1 hour and only needs water for the destaining process.
However, in acidic conditions, coomassie dye binds to the basic hydrophobic residues
of proteins as a result changes the colour from a dull red colour to an intense blue.
Coommasie can detect as little as 8-10ng per band of proteins or up to 25ng per band of
proteins. The chemicals needed for this staining would be the coomassie dye, an initial
wash step to remove excess SDS, as this would disrupt the binding of the dye, after
staining  The wavelengths used in staining 

Reagents and Materials: 

 4mM glucose solution
 Distilled water
 Sucrose solution 
 Deionized water 
 Acetate buffer 
 Nelson’s reagent 
 Arsenomolybdate reagent 
 Urea solution 
 Alcohol 
 0.1% bromophenol-blue solution 
 Ice
 Glycerol 
 12.5% TCA solution 
 Gel for SDS-PAGE 
 Coomassie blue dye 
Method: 
A.        The Effect of Enzyme Concentration on Initial velocity
 
Follow the procedure for the assay of enzyme activity as outlined in Table 4. The steps are
written below.
 
A  Nelson’s calibration curve was prepared as on outlined below and synchronise with 
samples;
      A calibration curve using 4mM glucose was prepared. Into 9 test tubes respectively, 0,
0.05, 0.05, 0.1, 0.15, 0.2, 0.2, 0.25 and 0.3mL of the standard glucose solution, were
pipetted , then all the volumes were made up to 1.0mL with distilled water. A series of 10
tubes were set up, where tubes 1 and 2 were the glucose and sucrose blanks respectively and
tube 3 was the glucose standard. A 1 in 1000 dilution of fraction 4 was prepared using cold,
deionized water. To tubes 4–9, the following volumes of diluted fraction 4: – 0.0, 0.02, 0.05,
0.1, 0.2, 0.4 and 0.6mL, were added. Then 0.2mL of 0.2M Acetate buffer of pH 4.5 were
added to each tube and brought the volumes to 0.8mL per tube with deionized water. Tube 10
is the zero-time control. It was prepared exactly as tube 9, however, 1mL of Nelson’s reagent
was added before adding the 0.2mL of sucrose (thus preventing any enzymatic formation of
reducing sugar).The reaction was started by adding 0.2mL of 0.5M sucrose and incubate for
EXACTLY 10 minutes at room temperature. The reaction was ceased by adding 1mL of
Nelson’s reagent to each tube except tube 10, and proceeded with Nelson’s assay for
reducing sugars as outlined above.The tubes were mixed well and placed in a boiling water
bath for EXACTLY 20 minutes. The tubes were left to cool to room temperature and then
1mL of the arsenomolybdate reagent was added, it was then mixed well on a vortex mixer
and left standing at room temperature for 5 minutes. Next 7mL of distilled water was added
to all tubes, mixed well on a vortex mixer and the absorbance at 510nm were read using tube
#1 as the blank for tubes 4 – 9. The number of units of enzyme activity per mL of original
fraction 4 using the results from this experiment (A), were calculated.The number of
micromoles of reducing sugar formed per minute (v) against the amount of enzyme (in mg
protein as determined for fraction 4 by the protein assay did previously), were plotted.
 
 
 
B.    Effect of Incubation time on Product Formation
 
          The enzyme (i.e. fraction 4) was diluted so that 0.1mL will yield 1μmol/min under
normal assay conditions, (i.e., 0.2mL of 0.5M sucrose, 0.2mL of 0.2M acetate buffer, pH 4.5,
in a final volume of 1mL and an incubation period of 10 minutes). The stock sucrose was
diluted 1:25 with deionized water. Then 12 tubes were set up for the assay of enzyme activity
as shown in Table 5. Tube 10 was the control without enzyme and tubes 11 and 12 are a
check on the standard curve. Tube 1 was the blank for tubes 2 –10 and tube 11 is the blank
for tube 12. The micromoles of reducing sugar formed as a function of incubation time (min),
were plotted.

C   The Effect of Substrate Concentration on Enzyme Activity 

A Michaelis-Menten curve was constructed as follows
 
0.2mL of 0.2M acetate buffer, pH 4.5, were added to ten test tubes. The following volumes of
0.5M sucrose were added to each respective tube: - 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.2
and 0.4mL. Tubes 9 and 10 are included to account for non-enzymatic hydrolysis of sucrose
(i.e. they are zero-time controls, therefore, 1mL of Nelson’s reagent must be added to tubes 9
and 10 BEFORE the start of the reaction). The volumes of each tube were brought to 0.9mL
by adding deionized water. Two more tubes labeled 11 and 12 were prepared. To tube 11
1.0mL of deionized water was added. To tube 12 0.8mL of deionized water and 0.2mL of
standard glucose solution was added. Fraction 4 was diluted, 1 in 1000 with cold, deionized
water.
 The contents of the tubes were then mixed thoroughly on a vortex mixer.  The reactions in
tubes 1 – 10 were started by adding 0.1mL of diluted Fraction 4. The amount of enzyme
added should be non-limiting i.e. about 2/3 up the linear portion of the enzyme concentration
curve. If this calls for more than 0.1mL of enzyme solution, the volume adjustment in step 3
should change accordingly e.g. if 0.2mL of enzyme is required, adjust the volumes to 0.8mL
with deionized water. Incubate all tubes for EXACTLY 10 minutes and stop the reaction by
adding 1mL of Nelson’s reagent to tubes 1 – 8 and tubes 11 and 12. The tubes were placed in
a boiling water bath for 20 minutes and continue with Nelson’s procedure for reducing sugars
(i.e. allow to cool, add 1mL arsenomolybdate solution and mix. It was left to stand for 5
minutes, add 7mL deionized water, mix well and read absorbance at 510nm). The absorbance
at 510nm were read and v (μmoles of reducing sugar/min) and [S], the concentration of
sucrose in each tube (mM), were calculated. Graphs of rate vs. substrate concentration
(Michaelis-Menten curve) and 1/v vs. 1/[S] (Lineweaver-Burke plot) were generated. K and m

V values were calculated and reported.

max

D     Inhibition by Urea
         The effect of the inhibitor is determined by the set up 8 test tubes with increasing
amount of sucrose as shown in Table 7 (0.0mL – 0.4mL substrate), 0.25mL of a 4M Urea
solution were added and the volumes were adjusted  appropriately to 0.9ml with deionized
water. To correct for non-enzymatic hydrolysis, 2 tubes similar to 9 and 10 in the table but
also containing 0.25mL of 4M urea, were added.It was ensured that the amount of water
added gives a final volume of 0.9mL. 1mL of Nelson’s reagent was added before adding the
enzyme. The same amount of enzyme you used in experiment C, was added. The reaction
was started by adding 0.1mL enzyme (or the volume used in experiment C). Incubate the
tubes for exactly 10minutes. The reaction was then stopped  by adding 1mL of Nelson’s
reagent and proceeding with Nelson’s procedure for reducing sugars. The sucrose
concentration in each sample was calculated.   Graphs representing v (μmoles of reducing
sugar produced/min.) as a function of [S] substrate concentration in the presence and absence
of urea were plotted. Also a the Lineweaver-Burke plot of 1/v vs. 1/[S] was done. The
apparent V and K in the presence and absence of urea, was calculated. Based on your
max m

results (graphs and calculations), the type of inhibition observed was determined. 
 
E     Polyacrylamide Gel Electrophoresis using fractions 1 - 4
 
Preparation of the samples to be applied to the gels
 
     The amount of protein (as determined in session 3) in the crude, alcohol and heat fractions
and then dilute the fractions to a concentration of  1mg/mL. The column fraction must be
concentrated by ultrafiltration, were calculated. The for each of the above fractions the
following in micro-centrifuge tubes were prepared: 50μL diluted protein, 25μL 50% glycerol,
20μL upper reservoir buffer and 5μL 0.1% bromophenol-blue solution. Keep the prepared
fractions on ice.
 
       The Fraction 4 protein solutions were concentrated by use of a filtering system designed
for micro-centrifugation. The “ultraspin” tubes consist of an insert fitted with a membrane
that has a molecular weight cut off level of 100,000 Da. This insert fits into a micro-
centrifuge tube in which the effluent is collected upon centrifugation. The concentrated
protein solution is retained in the insert. This system can accommodate 0.45ml of solution.
 
Procedure for concentrating Fraction 4 using the “ultraspin” system
 
         The insert was placed into the micro-centrifuge tube and 0.40ml of fraction 4 was
pipetted into the insert. The cap was pressed into place and spun at 5000 rpm for 5minutes.
The effluent (i.e. the solution that is in the micro-centrifuge tube)was decanted into a test
tube. Another 0.40ml of fraction 4 was pipetted into the insert and spun again. The effluent
was decanted and the process was repeated spinning this time for 5 minutes only. The
concentrated protein solution was removed without disturbing the membrane by gently
aspirating with an automatic pipette and transfer to a clean micro-centrifuge tube.
Approximately 50μl of this concentrated sample was needed. Then 50μL of fraction 4 was
transferred to a clean micro-centrifuge tube and add 25μL 50% glycerol; 20μL upper
reservoir buffer and 5μL 0.1% bromophenol-blue solution. The prepared fractions were kept
on ice. A blank reaction tube was prepared, which contained 50μL deionized water, 25μL
50% glycerol; 20μL upper reservoir buffer and 5μL 0.1% bromophenol-blue solution. Next,
40μL of each fraction was loaded onto the gel. The upper reservoir buffer was poured over
the gel and cover.  The power supply was switched and set to 30mAmp and run for 1.5 -2
hours. After the end of the run, the power supply was switched off and unplugged. The gel
was carefully removed and the distance from the top of the gel to the dye front was measured.
The gel was immersed  in 12.5% TCA solution for 30 minutes to fix the proteins in the gel.
The TCA from around the gel was removed using a pasteur pipette. The Coomassie blue stain
was added, and it ensured that the gel was completely submerged for 30 minutes. Then
remove the gels and place in the destaining solution for 3 – 5 hours.
 
 
 
 
 
 
 
 
 
 Results :

Table 1- Calibration curve for reducing sugars

 
Tube Vol. of glucose standard (mL) Abs. @ 510 μmol of reducing sugar
# nm

1 0.00 0.000 0.00

2 0.05 0.167 0.20

3 0.05 0.171 0.20

4 0.10 0.345 0.40

5 0.15 0.511 0.60

6 0.20 0.634 0.80

7 0.20 0.705 0.80

8 0.25 0.860 1.00

9 0.30 1.081 1.20

10 Fructose (4mM, 0.2mL) 0.000 0.80

11 Sucrose (4mM, 0.2mL) 0.167 0.80

           
 

Table 2
Additions SUCROSE GLUCOSE GLUCOSE FRACTION 4 ZERO TIME
BLANK BLANK STANDARD CONTROL
Tube # 1 2 3 4 5 6 7 8 9 10
Acetate 0.2 --- --- 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Buffer
Deionized 0.6 1.0 0.8 0.58 0.55 0.5 0.4 0.2 --- ---
Water
Volume of --- --- --- 0.02 0.05 0.1 0.2 0.4 0.6 0.6
Fraction 4
Volume of --- --- 0.2 --- --- --- --- --- --- ---
glucose
4mM
START REACTION BY ADDING SUBSTRATE
Vol. of 0.5M 0.2 --- --- 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Sucrose
Incubate all tubes for 10 minutes at room temperature. Stop the assay by addition of Nelson’s
reagent. VORTEX!
Nelson’s 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
reagent
Place the tubes in a boiling water bath and continue with the Nelson’s procedure for reducing
sugars. VORTEX!
Absorbance 0.134 0.025 0.528 0.212 0.340 0.486 0.686 0.994 0.145 0.052
at 510nm
μmol 0.00 0.00 0.80 0.08 0.20 0.40 0.80 1.60 2.40 2.40
reducing
sugar in 10
min.
μmol/min/mL

Avg.
μmol/min/mL
Units/mL
stock fraction