Annie Chen

3/22/2018

Module D

Background

The purpose of this experiment was to investigate enzyme kinetics and explore the different methods that
can be used to determine the rate of catalytic reactions and the properties of enzymes, as well as glucose
concentration in blood.

Enzymes are macromolecular biological catalysts that accelerate chemical reactions, turning substrate
into product. Enzymes are vital to human life, as almost all metabolic processes that occur in our cells
require enzyme catalysis to operate fast enough to sustain life. By lowering activation energy, enzymes
increase the reaction rates, sometimes millions of times faster. In order to do this, enzymes must bind
their substrate. Enzymes usually have very high specificity as to which substrates they can bind, achieved
through binding pockets customized for specific substrates by shape, charge, and
hydrophobic/hydrophilic properties.

The study of how enzymes bind substrates and turn them into products is called enzyme kinetics. One
quantitative theory of enzyme kinetics is what is known as Michaelis-Menten kinetics. In this theory, an
enzyme reaction has two stages: first, the substrate binds irreversibly to the enzyme, forming an enzyme-
substrate (ES) complex; second, the enzyme catalyzes the substrate and releases the product. Enzyme
rates are dependent on solution conditions and substrate concentration. Generally, the more substrate
there is, the faster the reaction. However, enzymes have a saturation limit – when all free enzymes are
bound to substrate to form the ES complex, the maximum rate of the reaction is reached (V max). There are
also other kinetic parameters, such as Km, which is the substrate concentration required for an enzyme to
reach one-half of its maximum reaction rate. An enzyme can have multiple K m that correspond to different
substrates. Another parameter, kcat, is the turnover number, which is the number of substrate molecules
handled by one active site per second. kcat/Km together can be used to express an enzyme’s efficiency and
since it reflects both affinity and catalytic ability, efficiency is a good way to compare enzymes against
other enzymes or different substrates. Theoretically, efficiency is limited to 10 9 M-1s-1, which means that
every collision of enzyme and substrate will result in the formation of product so the only thing that limits
the reaction would be the rate of diffusion. Only a few catalytically perfect enzymes can achieve this kind
of efficiency, and most enzymes do not come close.

Enzymes may be used to detect and quantify the presence of other substances in a solution. In a GO/HRP
glucose assay, sample glucose is converted to gluconolactone and hydrogen peroxide by glucose oxidase,
and hydrogen peroxide and ABTS are converted to ABTS+ and water by horseradish peroxidase.

By converting ABTS to ABTS+, the absorbance maximum shifts to about 420 nm which can easily be
measured by a spectrophotometer, and a glucose concentration can be determined. The end product is
also soluble and colors the solution green so it is easy to distinguish which solution has glucose and which
doesn’t.
 Annie Chen
3/22/2018

Chemical reactions can be either reversible or irreversible. In irreversible reactions, all of the reactants
get converted into products, and the products cannot go back to being reactants. The irreversible reaction
conducted in this experiment is the alkaline phosphatase reaction:

Alkaline phosphatase catalyzes the hydrolysis of esters in phosphoric acids in an alkaline environment.
The absorbance at 405 nm of the product p-nitrophenol can be measured to calculate its concentration
through Beer’s law. Because all reactants are turned into product, the final product concentration can
also be used to calculate the concentrations of the reactants. In this particular case, the mole ratio was
1:1 so the final concentration of p-nitrophenol was equal to the original concentration of p-
nitrophenylphosphate. Plotting the absorption values on a graph versus reaction time, the initial velocity
of the reaction can be estimated by the slope of a regression line through the beginning linear phase.
Once the absorption plateaus to a constant value, that indicates that all reactants have been turned into
product and the reaction has gone to completion. The absorbance at that point can be used to calculate
the initial concentration of pNPP through Beer’s Law. An abundance of Tris buffer was added to the
solution to make sure the pH (and thus the hydrogen ion concentration) stayed relatively constant.

If a reaction is not irreversible, it is some varying degree of reversible. That means the reaction proceeds
both forwards and backwards simultaneously until dynamic equilibrium is achieved; that is, until reactants
are formed at the same rate as products (k1 = k-1). The reversible reaction studied in this experiment was
the alcohol dehydrogenase reaction:

Alcohol dehydrogenase (ADH) catalyzes the conversion of ethanol to acetaldehyde through the oxidation
of NAD+ to NADH. NADH has an observable absorption maximum at 340 nm, so the absorption values
collected throughout the reaction can be used to calculate the concentration of reactants at equilibrium.
Although the mole ratios are also 1:1, the initial ethanol and NAD+ concentrations used in this experiment
were diluted from a stock concentration, so the dilution factor had to be accounted for when doing
calculations. All reversible reactions have an equilibrium constant, K, that depends on the Gibbs free
energy change of the reaction. The larger the free energy change, the larger Keq will be, and the reaction
will tend to favor the forward reaction making products.

Scatchard plots and Job’s method can be used to determine the protein-binding parameters of enzymes
in where two species are present. In this experiment, the two different methods were used to ultimately
find the average number of ANS binding sites per BSA molecule. Before constructing the Scatchard plot,
the measured absorbance is plotted against ANS concentration so that a curve is obtained. This first few
data points in this initial plot can be used to determine the B-factor (instrument response factor) of the
reaction by assuming that [ANS]bound is equal to [ANS]total at the beginning. The B-factor can be used to
calculate the concentration of bound ANS molecules, which can be used to solve for the concentration of
free ANS molecules. Once that information has been gathered, the Scatchard plot can be constructed by
plotting r (the ratio of bound ANS to BSA) divided by the concentration of free ANS against r of the
remaining data points (the points used to calculate the B-factor are omitted). The plot results in a linear
line of negative slope of equation:
 Annie Chen
3/22/2018

𝑟 𝑛 𝑟
= −
[ANS] 𝐾 𝐾
With slope equal to -1/Kd and the y-intercept equal to n/Kd.

In Job’s method, the reaction is conducted with mole fractions of ANS. Plotting absorbance at 470 nm
against the mole fractions results in a slightly parabolic-looking graph. Least-squares regression lines are
fitted to the front and back portions of the plot so that an intersection between the two lines is achieved.
The intersection is the ANS mole fraction at which the reaction is at maximum velocity. Since the mole
fraction of ANS is directly related to the mole fraction of BSA, the ANS mole fraction at maximum velocity
can be used to determine n.

Lineweaver-Burk and Hanes-Woolf plots can be used to determine the kinetic parameters of enzymes.
The Lineweaver-Burk plot plots the reciprocal of the reaction velocity against the reciprocal of the
substrate concentration. Subsequently, the slope is km/Vmax and the y-intercept is 1/Vmax. The Lineweaver-
Burk equation is as follows:
1 𝑘 1 1
= +
𝑉 𝑉 [𝑆] 𝑉
Thus, km can be determined by taking the negative reciprocal of the x-intercept, and Vmax can be
determined by taking the reciprocal of the y-intercept. The velocities of the reaction can be found on a
standard plot of product concentration versus time.

Like the Lineweaver-Burk plot, the Hanes-Woolf plot also transforms data into a linear relationship, but
with a slightly different equation:
[𝑆] [𝑆] 𝑘
= +
𝑉 𝑉 𝑉
In this graph, km is determined by the negative value of the x-intercept and Vmax is the reciprocal of the
slope. One disadvantage of the Hanes-Woolf plot is that both the x- and y-values are dependent on
substrate concentration. As a result, the correlation coefficient R is not applicable.

The enzyme studied in this experiment was wheat germ acid phosphatase (WGAP). It catalyzes the
hydrolysis of monoesters and anhydrides of phosphoric acid to produce inorganic phosphate.

Data 1

Alkaline Phosphatase (irreversible)

Time (s) A405 ε405 (M-1cm-1) [P] (mol/L)
0 0 1.8E4 0
30 0.191 1.8E4 1.06E-05
60 0.269 1.8E4 1.49E-05
90 0.298 1.8E4 1.66E-05
120 0.310 1.8E4 1.72E-05
150 0.315 1.8E4 1.75E-05
180 0.317 1.8E4 1.76E-05
210 0.318 1.8E4 1.77E-05
240 0.318 1.8E4 1.77E-05
 Annie Chen
3/22/2018

300 0.318 1.8E4 1.77E-05

Alcohol Dehydrogenase (reversible)

Time (s) A340 ε405 (M-1cm-1) [P] (mM)
0 0 6.22E3 0
30 0.131 6.22E3 2.11E-02
60 0.187 6.22E3 3.01E-02
90 0.219 6.22E3 3.52E-02
120 0.242 6.22E3 3.89E-02
150 0.261 6.22E3 4.20E-02
180 0.278 6.22E3 4.47E-02
210 0.291 6.22E3 4.68E-02
240 0.302 6.22E3 4.86E-02
300 0.320 6.22E3 5.14E-02
360 0.334 6.22E3 5.37E-02
420 0.344 6.22E3 5.53E-02
480 0.352 6.22E3 5.66E-02
540 0.358 6.22E3 5.76E-02
600 0.363 6.22E3 5.84E-02
720 0.370 6.22E3 5.95E-02

.
[𝑃] = = = 2.11 × 10 M = 2.11 × 10 mM
( . × M cm )( cm)

Data 2

Tube 2 A415: 1.355

Tube 3 A415: 0.295

Without invertase: no color change observed

With invertase: drop turned blue-green when sucrose added

Data 3

Binding Curve Analysis

Tube [BSA] [ANS] Buffer BSA ANS Total F470 [ANS]bound [ANS]free r r/[ANS]free
(μM) (μM) (μL) (μL) (μL) (μL) (μM) (μM) (μM-1)
1 10 2 987 1000 13 2000 135.265 x x x x
2 10 5 967 1000 33 2000 185.800 x x x x
3 10 10 933 1000 67 2000 322.496 x x x x
4 10 15 900 1000 100 2000 457.726 12.873 2.127 1.287 0.605
 Annie Chen
3/22/2018

5 10 20 867 1000 133 2000 548.307 15.421 4.579 1.542 0.337

6 10 25 833 1000 167 2000 643.456 18.097 6.903 1.810 0.262
7 10 30 800 1000 200 2000 722.012 20.306 9.694 2.031 0.209
8 10 40 733 1000 267 2000 883.200 24.840 15.160 2.484 0.164
9 10 60 600 1000 400 2000 767.575 21.588 38.412 2.159 0.056
F F
𝐵= = = 35.556 μM
[ANS]bound [ANS]total

F .
[ANS]bound = = = 12.873 μM
. μM

[ANS]free = [ANS]total – [ANS]bound = 15 μM – 12.873 μM = 2.127 μM

[ANS]bound 12.873 μM
𝑟= = = 1.287
[BSA] 10 μM

Job’s Method
Tube XANS ANS (mL) BSA (mL) Total (mL) F470
1 0.2 0.4 1.6 2.0 157.701
2 0.3 0.6 1.4 2.0 305.460
3 0.4 0.8 1.2 2.0 214.004
4 0.5 1.0 1.0 2.0 274.976
5 0.55 1.1 0.9 2.0 283.724
6 0.65 1.3 0.7 2.0 311.399
7 0.7 1.4 0.6 2.0 310.735
8 0.75 1.5 0.5 2.0 333.420
9 0.8 1.6 0.4 2.0 276.804
10 0.9 1.8 0.2 2.0 223.696

Data 4

Tube A405 Min. at H2O (μL) pNPP (μL) Acetate KOH ε405 (M- [P] (M)
1
37° (μL) (μL) cm-1)
1 0.024 0 470 20 500 500 1.8E04 1.33E-06
2 0.156 3 470 20 500 500 1.8E04 8.67E-06
3 0.281 6 470 20 500 500 1.8E04 1.56E-05
4 0.689 9 470 20 500 500 1.8E04 3.83E-05
5 0.018 0 475 15 500 500 1.8E04 1.00E-06
6 0.124 3 475 15 500 500 1.8E04 6.89E-06
7 0.267 6 475 15 500 500 1.8E04 1.48E-05
8 0.413 9 475 15 500 500 1.8E04 2.29E-05
9 0.022 0 480 10 500 500 1.8E04 1.22E-06
10 0.148 3 480 10 500 500 1.8E04 8.22E-06
11 0.315 6 480 10 500 500 1.8E04 1.75E-05
12 0.277 9 480 10 500 500 1.8E04 1.54E-05
13 0.045 0 485 5 500 500 1.8E04 2.50E-06
14 0.072 3 485 5 500 500 1.8E04 4.00E-06
15 0.138 6 485 5 500 500 1.8E04 7.67E-06
16 0.209 9 485 5 500 500 1.8E04 1.16E-05
 Annie Chen
3/22/2018

Data Analysis 1

Irreversible Reaction
0.00003
y = 3.54E-07x
0.000025

0.00002
[P] (mol/L)

0.000015

0.00001

0.000005

0
0 50 100 150 200 250 300 350 400
Time (s)

[ ] . ×
Alkaline phosphatase vo: = = 𝟑. 𝟓𝟒 × 𝟏𝟎 𝟕
Ms 𝟏

5 μL pNPP
= 0.005% = 0.00005
1000 μL total solution
1.77 × 10 M = 0.00005[pNPP]o

[pNPP]o: 0.354 M
[ ] . ×
NADH vo: = = 𝟕. 𝟎𝟐 × 𝟏𝟎 𝟕
Ms 𝟏

Sources of error: when mixing the solution by pipetting a small amount of solution was left on the pipette
tip; sometimes the reaction would be stopped late because there wasn’t enough time to prepare the
materials (especially in the 30 second intervals)
 Annie Chen
3/22/2018

Reversible Reaction
0.00007

0.00006

0.00005
y = 7.02E-07x
[P] (mol/L)

0.00004

0.00003

0.00002

0.00001

0
0 100 200 300 400 500 600 700 800
Time (s)

[CH3CHO]eq = [NADH]eq = 5.95 × 10 M = 5.95 × 10 mM

[EtOH]stock ( mM)( . mL)
[EtOH]o = = = 21.25 mM
( mL)

[NAD]stock ( . mM)( . mL)

[NAD+]o = = = 0.0825 mM
( mL)

[H+]o = [H+]eq = 10 M = 10 mM

[EtOH]eq = [EtOH]o – [acetaldehyde]eq = 21.25 mM − 0.0595 mM = 𝟐𝟏. 𝟏𝟗 mM

[NAD+]eq = [NAD+]o – [NADH]eq = 0.0825 mM − 0.0595 mM = 𝟎. 𝟎𝟐𝟑 mM

[CH CHO]eq [NADH]eq [H ]eq (5.95 × 10 mM)(5.95 ×10 mM)(10 mM)

k eq = =
[EtOH]eq [NAD ]eq (21.19 mM)(0.023 mM)
𝟖
= 𝟕. 𝟐𝟔 × 𝟏𝟎 mM
Sources of error: sometimes the reaction would be stopped late because there wasn’t enough time to
prepare the materials (especially in the 30 second intervals)

Data Analysis 3
 Annie Chen
3/22/2018

Binding Curve Analysis

1000
900 y = 35.556x + 24.057
800
700
600
F470

500
400
300
200
100
0
0 10 20 30 40 50 60 70
[ANS]total (μM)

B-factor: 35.556 μM-1

Scatchard Plot
1.200

1.000

0.800
y = -0.3781x + 0.9852
r/[ANS]free

0.600

0.400

0.200

0.000
-0.5 0 0.5 1 1.5 2 2.5 3
-0.200
r

KD = -1/m = -1/(-0.378 μM) = 2.645 μM

n = K D × 𝑦 = (2.645 μM)(0.9852 μM ) = 2.606

Sources of error: inaccurate pipetting due to the fact that different sized pipettors have different levels
of precision so sometimes multiple pipettors had to be used to achieve one volume of solution. For
example, 166.7 μL would be made with 100 μL, 60 μL, and 6.7 μL in an effort to avoid the low end of the
pipettors. This method, however, is difficult to keep consistent since different pipettors need to be
adjusted for each sample depending on the target volume.
 Annie Chen
3/22/2018

Job's Method
500
y = 434.46x + 59.667
450
R² = 0.7527
400
350
300
F470

250 y = -164.2x + 437.39

200 R² = 0.36
150
100
50
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
XANS

−164.2𝑥 + 437.39 = 458.78𝑥 + 53.911

XANS at intersection = 0.631
[ANS] 𝑋ANS 𝑋ANS 0.631
𝒏= = = = = 𝟏. 𝟕𝟏𝟎
[BSA] 𝑋BSA 1 − 𝑋ANS 1 − 0.631
Sources of error: inaccurate pipetting of small volumes with 1000 μL pipettor, bubbles remaining in the
solution when taking measurements in the fluorometer

Data Analysis 4
 Annie Chen
3/22/2018

vo 1/[S] (mM- 1/vo [S] (mM) [S]/vo

1
(mM/min) )
S1 3.93E-03 1.5 2.54E02 0.666667 1.70E05
S2 2.46E-03 2 4.07E02 0.5 2.03E05
S3 1.73E-03 3 5.78E02 0.333333 1.93E05
S4 1.03E-03 6 9.71E02 0.166667 1.62E05
 Annie Chen
3/22/2018

Lineweaver-Burke Plot
1.20E+03
y = 151.69x + 78.462
1.00E+03

8.00E+02
1/vo (min/mM)

6.00E+02

4.00E+02

2.00E+02

0.00E+00
0 1 2 3 4 5 6 7 8
1/[S] (mM-1)

Km = 1.93 mM

Vmax = 1.27 x 10-2 mM/min

Hanes Plot
2.50E+02

2.00E+02
y = 1.23E+02x + 1.45E+02
[S]/vo (min)

1.50E+02

1.00E+02

5.00E+01

0.00E+00
-0.2 0 0.2 0.4 0.6 0.8
[S] (mM)

Km = 1.18 mM

Vmax = 8.13 x 10-3 mM/min

Average Km = 1.56 mM ± 0.375 mM

 Annie Chen
3/22/2018

Average Vmax = 1.04 x 10-2 mM/min ± 2.29 x 10-3 mM/min

58 kDa = 58000 g/mol

mg
[E]t = × × × × = 𝟎. 𝟎𝟎𝟎𝟏𝟕𝟐 𝐦𝐦𝐨𝐥/𝐋
mL

Vmax = kcat[E]t

Vmax 1.04 × 10 mM/min 𝟏 𝟏

k cat = = = 𝟔𝟎. 𝟓 𝐦𝐢𝐧 = 𝟏. 𝟎𝟏 𝐬
[E]t 1.72 × 10 mM
k cat .
Eefficiency = = = 𝟑𝟖. 𝟖 𝐦𝐌 𝟏
𝐦𝐢𝐧 𝟏
= 𝟔𝟒𝟕 𝐌 𝟏
𝐬 𝟏
km . mM

Sources of error: small concentrations are most prone to pipetting error and those data points are the
ones that have the most influence in the slope of double reciprocal plots like the Lineweaver-Burk plot.
Also, it is almost impossible for each reaction to be started and stopped at exactly the same time every
repetition, even if all 4 of the assays were run one at a time. There will always be a little discrepancy in
the time that the reactions are allowed to run, and those discrepancies are amplified by the linear
transformation in the plots.

Discussion Questions 2

The normal blood glucose range for humans is 4.4 to 6.1 mM, so 5 mM is about in the middle of that range.
Based on the experimental data, blood serum components drastically decrease the absorbance of the
reaction mixture. In clinical use, this would be cause for concern. One possible solution is presented by
Segawa, et al. 1992, where the GO reaction is coupled with a fluorescein-chemiluminescent (FL-CL)
reaction instead of ABTS. The method was said to “not [be] subject to any interference from components
normally present in serum.”

The purpose of incubating the sucrose solution without invertase at 55°C is to make sure the results
observed are due to the addition of invertase and not just because of heating the solution. The treatment
with invertase was successful based on the color change of the ABTS drops. The drop with invertase turned
blue-green and the drop without invertase did not change color.

If the product is intended for consumer use it has to be able to very clearly distinguish with the naked eye
between the detected levels of glucose. There must be a set gradient of color that corresponds to ranges
of glucose levels so that consumers can clearly and efficiently compare and identify their own glucose
level.

Discussion Questions 3

The two n-values were fairly different, with n = 2.606 for the Scatchard Plot and n = 1.710 for Job’s method.
The difference may be due to the fact that in Scatchard analysis the value of X (bound) is used to calculate
Y (bound/free) with bound ligand being used in both values, and this violates the assumption of linear
 Annie Chen
3/22/2018

regression that all uncertainty is in Y while X is known precisely. Distortion of data during linear
transformation can cause the calculated n-value to vary.

The data for Job’s method is lower quality, as the selected data points are not particularly linear (R 2 value
is low), so the difference can also probably be attributed to experimental error.

Discussion Questions 4

Based on the calculations, WGAP seems to be less efficient than the typical enzyme. The average efficiency
of enzymes is about 105 M-1s-1, while the calculated value for WGAP is only 647 M-1s-1. Since the binding
between enzyme and substrate is the rate-determining step in a catalytic reaction, it means that WGAP is
less efficient than average at binding substrate. The smaller k m is, the better the enzyme is at binding the
substrate, and the km of WGAP is relatively large compared to the average enzyme (km = 0.1 mM compared
to WGAP at km = 1.56 mM). Theoretically, WGAP should be only slightly less efficient than the average
enzyme at an efficiency of 104 M-1s-1.