C O LO R I M E T R I C E N Z Y M E A S S AY O F

G LU C O S E OX I DA S E A N D
D E T E R M I N AT I O N O F M O L E C U L A R
W E I G H T O F G LU C O S E OX I DA S E
E N Z Y M E T H R O U G H G E L P E R M E AT I O N
C H R O M AT O G RA P H Y
Patrick E. Smith Jr. and Jack Wardale
SETON HILL UNIVERSITY SCH 326 01

MARCH 31, 2016

ABSTRACT:

In part A of this experiment, the primary purpose was to determine the specificity of
the enzyme Glucose Oxidase (GOx) for its substrate beta-D-Glucose by determining the
amount of glucose in a series of standards. This was achieved by a coupled reaction that
acted as a biosensor, using the absorbance data retrieved from the colorimetric enzyme
assay data derived from the oxidation of ferrocyanide to ferricyanide to conduct
spectrophotometric analysis. This experiment successfully identified that the Gatorade
sample had X mM glucose and that the super sugar solution A contained the added
glucose. It also alluded to the high specificity of GOx to glucose since the Super Sugar B
solutions and the undigested sugar samples produced a low concentration of glucose
using this method.
The purpose of Part B of this experiment was to determine the molecular weight of
GOx through gel permeation chromatography by using Blue- Dextran 2000, Hemoglobin,
and DNP-Glycine as standards to create a calibration curve that produced an equation in
order to calculate the molecular weight based on the retention volume recorded from the
experiment. The retention volume for GOx determined from the analysis was 10.56mL.
This value was then used to calculate the molecular weight of GOx, which was 127,270 Da
producing a 20.46% error based on the 160,000 Da literature value for the molecular
weight of GOx found.

INTRODUCTION:

The objective of the first experiment is to experimentally test various sugar samples
and a commercially available sports drink using spectrophotometry to determine the
concentration of glucose present. This is achieved by a coupled reaction using the
enzymes glucose oxidase and horseradish peroxidase. Glucose oxidase reacts with betaD-Glucose to produce hydrogen peroxide and beta-D-gluconolactone. This hydrogen

peroxide is a reactant in the second reaction, which causes the oxidation of ferrocyanide
to ferricyanide, creating a yellow color that can be spectrophotometrically analyzed in
order for the concentration of glucose to be quantified. The secondary objective is to
determine what kind of specificity glucose oxidase exhibits by measuring the absorbance
of different sugar samples using spectrophotometry at 420 nm, which is the wavelength
max for ferricyanide. The final objective of the first experiment is to deduce which of the
super sugar solutions contains added glucose.
Glucose in solution is often quite hard to quantify as an analyte as part of a complex
matrix. In order to experimentally determine the glucose concentration therefore, an
alternative method had to be used. The glucose oxidase product of hydrogen peroxide is
used to allow the oxidation reaction of ferrocyanide to ferricyanide to occur, creating a
yellow compound that can be measured by a spectrophotometer for absorbance. The
higher the absorbance recorded, the more ferricyanide in solution, since absorbance is
proportional to concentration due to the Beer-Lambert law. The assay is therefore an
indirect measure of the amount of glucose in a solution because the reaction does not
directly measure glucose, but instead it measures the amount of ferricyanide produced.
Since this mechanism has a stoichiometric ratio of 1:1:1:1, the concentration of
ferricyanide is equivalent to the concentration of beta D- glucose present. This is
important because this is a colorimetric assay and beta-D-glucose does not produce a
color that can be measured in absorbance. The proportionality between ferricyanide and
beta-D-glucose allows for the absorbance of ferricyanide to be used to calculate the
concentration of ferricyanide, which is in turn is the concentration of beta-D-glucose.
Though this assay does not directly measure glucose concentration, it should still
accurately be able to determine the concentration of glucose based on the stoichiometric
ratio of both enzyme reactions. The absorbance obtained from the samples will be
compared to a standard curve of known concentrations of glucose.
Glucose oxidase has a unique specificity for -D-glucose and does not react with the
alpha stereoisomer (Tao et al., 2009). Therefore, it was assumed that all of the glucose
present in the sports drink was in this stereoisomer and the type of specificity it exhibits is
stereospecific. It was hypothesized therefore that if the sugar samples did not have
glucose in the -D isomer, the yellow product would not be formed. Glucose oxidase is an
oxidoreductase enzyme, one that can catalyze oxidation and reduction reactions
(BRENDA). The Glucose oxidase mechanism involves catalyzing the oxidation of an alcohol
group to a ketone, the alcohol being the electron donor and oxygen being the electron
acceptor to form hydrogen peroxide (Weibels & Brights, 1971). The structure of glucose
oxidase involves all three forms of secondary structure, namely beta sheets, alpha helices
and random coils and exists as a dimer with two molecules of flavin adenine dinucleotide
(FAD) tightly bound per dimer. The FAD stabilizes glucose oxidases tertiary structure
(Raba & Mottola, 1995).
The other part of the coupled reaction also uses an oxidoreductase enzyme in
horseradish peroxidase. The second reaction involves the oxidation of ferrocyanide to
produce the yellow compound ferricyanide, this time the hydrogen peroxide acting as the
electron acceptor and the ferrocyanide being the electron donor (BRENDA). An enzyme
with six subunits, horseradish peroxidases secondary structure consists of all three
secondary structures also, and has two different ligands, a calcium ion and protoporphyrin
IX containing heme (RCSB).
The coupled reaction can be used as a biosensor, since the two enzymes are used
in order to detect an analyte of interest, in this case glucose. Biosensors play all kinds of
roles in industry. Glucose oxidase and horseradish peroxidase have recently been used in
the electrochemical determination of glucose using a nanomaterial graphene transducer
and a polytoluidine blue film (Wang et al., 2015).

The objective of the second experiment is to determine the molecular weight of

glucose oxidase using molecular sieve chromatography. This would be achieved by using
the known molecular weights of blue dextran, hemoglobin and DNP-Glycine in order to
create a calibration curve of the log of the molecular weight against the retention volume
generated from the column.
Molecular sieve chromatography is a method of separating substances based on
molecular weight. It contains two phases, the stationary phase and the mobile phase. The
stationary phase in this experiment is Sephadex gel G 150 slurry. Sephadex is the
trademark name for cross-linked dextran gel (Andrews, 1964). In these cross-linked
strands, there are crevices and pores in which smaller molecules can sit, however larger
molecules cannot. The smaller molecules therefore take longer to elute from the column
than the larger molecules. The mobile phase on the other hand was a 0.05 M phosphate
buffer, used to help elute the molecules out of the column.
The three known molecular weights of colored compounds, bovine hemoglobin, blue
dextran 2000 and DNP-Glycine would come out of the column at different times, known as
the retention volume. This retention volume would be the volume in which took the
brightest color to be observed for each of the three compounds. These compounds would
then be used to plot a standard curve and then glucose oxidase would also be ran through
the column in order to determine its molecular weight. This value can then be compared
to literature values for the purpose of accuracy.

EXPERIMENTALMETHODS:

200 L of a Gatorade Fruit Punch Low Calorie sports drink was pipetted into a 10
mL volumetric flask and made up to the mark using distilled water. The standard and
unknown solutions were prepared using a micropipette as shown in Table 1 and Table 2
from left to right, except for the enzyme mix, which was added only once all standards
had been made. Once the enzyme mix was added, the enzyme solutions were transferred
to test tubes, vortexed briefly before being placed in a 32.5 oC water bath for 46 minutes.
Once calibrated, the SpectroVis spectrophotometer was set to 419. 6 nm and used to
record the absorbance of each of the heated samples three times.
For the second part of the experiment, the column was first prepared by packing 30
mL of Sephadex G150 slurry into the column using portions of 0.05 M phosphate buffer.
Once the gel was packed, a graduated cylinder was placed under the column and more
buffer solution was added until it just covered the gel and then 1 mL of standard dye
mixture was added. The stopcock was opened, allowing the buffer to drain out and it was
closed once the buffer just covered the gel layer. The buffer was added and ran through
the gel until the dye was an inch from the bottom of the column, at which point the
graduated cylinder was replaced with a test tube that had been marked 1 mL from the
bottom of the tube. The process of adding buffer continued, and the test tubes were
switched out every 1 mL interval until all the dye had ran out of the column. The most
vibrant color of each dye was used to obtain the retention volume by adding the prior
volumes to a graduated cylinder. To determine the molecular weight of glucose oxidase, 1
mL of a glucose oxidase solution was added to the same column in the same notion as the
standard dye mixture and the retention volume was again recorded in a graduated
cylinder once the most vibrant color had been chosen from the test tubes. This retention
volume was compared to the calibration curve produced by the retention volumes of Bluedextran 2000, hemoglobin and DNP-glycine as witnessed in Figure 2 in order to find out
the experimental molecular weight.

RESULTS:
4A DATA:
TABLE 1. PREPARATION OF GLUCOSE STANDARD SOLUTIONS FOR SPECTROPHOTOMETRIC ENZYME
ASSAY INVOLVING A 0.05 M PHOSPHATE BUFFER (ML), A 0.0625 M (ML), A 1 MM GLUCOSE SOLUTION
(ML) AND AN ENZYME MIX (ML).

0.0625 M
Ferrocyanide
Solution (mL)
1.0
1.0
1.0
1.0
1.0
1.0

0.05 M Phosphate
Buffer (pH 6.5) (mL)
3.5
3.0
2.5
2.0
1.5
1.0

1 mM Glucose
Solution (mL)

Enzyme Mix
(mL)

0.0
0.5
1.0
1.5
2.0
2.5

0.5
0.5
0.5
0.5
0.5
0.5

TABLE 2 . PREPARATION OF UNKNOWN SOLUTIONS FOR SPECTROPHOTOMETRIC ENZYME ASSAY

INVOLVING A 0.05 M PHOSPHATE BUFFER (ML), 0.065 M FERROCYANIDE SOLUTION (ML), AN
UNKNOWN SOLUTION (ML) AND AN ENZYME MIX (ML).

Sample
Sports Drink
Dilution 1
Sports Drink
Dilution 2
Undigested Sugar
Digested Sugar
Dilution 1
Digested Sugar
Dilution 2
Super Sugar A
Dilution 1
Super Sugar A
Dilution 2
Super Sugar B
Dilution 1
Super Sugar B
Dilution 2

0.05 M
Phosphate
Buffer (pH 6.5)
(mL)

0.065 M
Ferrocyanide
Solution (mL)

3.00

1.00

3.25

1.00

3.00

1.00

3.25

1.00

3.00

1.00

3.00

1.00

2.50

1.00

3.00

1.00

2.50

1.00

Unknown
Solutions (mL)
0.50
(Sports Drink)
0.25
(Sports Drink)
0.50
(Undigested Sugar)
0.25
(Digested Sugar)
0.50
(Digested Sugar)
0.50
(Super Sugar A)
1.00
(Super Sugar A)
0.50
(Super Sugar B)
1.00
(Super Sugar B)

Enzyme Mix
(mL)
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50

TABLE 3 .THE ABSORBANCE AND AVERAGE ABSORBANCE FOR THE 6 STANDARDS AT 419.6 NM TAKEN
FROM THE SPECTROVIS.

Concentration
of glucose
standard (mM)

Absorbance 1

Absorbance 2

Absorbance 3

Average
absorbance

0.0

0.000

0.000

0.000

0.000

0.1

0.214

0.208

0.236

0.219

0.2

0.385

0.384

0.390

0.386

0.3

0.527

0.575

0.560

0.554

0.4

0.761

0.757

0.756

0.758

0.5

0.786

0.826

0.833

0.815

TABLE 4. ABSORBANCES FOR THE UNKNOWN GLUCOSE CONCENTRATION SAMPLES AT 419.6

NM TAKEN FROM THE SPECTROVIS

Sample

Average

Absorbance 1

Absorbance 2

Absorbance 3

0.049

0.053

0.044

0.049

0.030

0.010

0.021

0.020

0.060

0.058

0.074

0.064

0.008

0.010

0.007

0.008

0.515

0.514

0.514

0.514

0.118

0.118

0.125

0.120

Absorbance

Sports Drink
Dilution 1
Sports Drink
Dilution 2
Undigested Sugar
Digested
Sugar
Dilution 1
Digested
Sugar
Dilution 2
Super Sugar A
Dilution 1

Super Sugar A
Dilution 2

0.239

0.250

0.231

0.240

0.058

0.063

0.048

0.056

0.063

0.077

0.076

0.072

Super Sugar B
Dilution 1
Super Sugar B
Dilution 2

Standard curve for Glucose Standards of Concentration vs. Absorbance @ 419.6nm

f(x) = 1.85x + 0.01
R = 1

FIGURE 1. STANDARD CURVE OF GLUCOSE STANDARDS FEATURING ABSORBANCE @ 419.6 NM VS

CONCENTRATION USED AS REFERENCE SAMPLES IN ORDER TO GENERATE AN EQUATION TO USE
SPECTROPHOTOMETRIC ANALYSIS TO INSERT ABSORBANCE READINGS TO FIND GLUCOSE
CONCENTRATIONS IN SAMPLES OF UNKNOWN GLUCOSE CONCENTRATION.

Equation: Y=1.851x + 0.0132 --> A=1.851c + 0.0132

Sports Drink 1 Test Tube Concentration Calculation
A=1.851c + 0.0132
0.049= 1.851c +0.0132
0.0358= 1.851c
c=0.0193
TABLE 5. VALUES FOR CONCENTRATION OF GLUCOSE IN EACH TEST TUBE CALCULATED USING THE
ABOVE EQUATION BASED ON THE STANDARD CURVE PRODUCED FROM THE 5 STANDARD GLUCOSE
SAMPLES SHOWN IN FIGURE 1.

Test Tube
Unknown
Samples

Solution
Absorbance

Glucose
Concentration
(mM)

Sports Drink 1

0.049

0.0193

Sports Drink 2

0.020

0.00367

0.064

0.0274

0.008

-0.0028

0.514

0.271

Super Sugar A1

0.120

0.0577

Super Sugar A2

0.240

0.123

Super Sugar B1

0.056

0.0231

Super Sugar B2

0.072

0.0318

Undigested
Sugar
Digested Sugar
1
Digested Sugar
2

Sports Drink 1
Initial Diluted Solution Glucose concentration
C1V1=C2V2
C1(0.5mL)=(0. 0193mM)(5mL)
C1= .193mM
Original Solution Glucose Concentration
C1(0.2mL)=(0.193 mM)(10mL)
C1= 9.65mM
Undigested Sugar
Initial Diluted Solution Glucose concentration
C1V1=C2V2
C1(0.5mL)=(0. 0274mM)(5mL)

C1= .274mM
Original Solution Glucose Concentration
C1(1mL)=(.274mM)(50mL)
C1= 13.7mM
TABLE 6. VALUES FOR CONCENTRATION OF GLUCOSE IN EACH TEST TUBE, EACH INITIAL DILUTION
AND EACH ORIGINAL SOLUTION FOR EACH SAMPLE OF UNKNOWN GLUCOSE CONCENTRATION
CALCULATED USING THE CALCULATIONS ABOVE.

Initial Diluted
Test Tube Solution
Original Solution
Unknown
Solution
Glucose Concentrati
Glucose Concentrati
Samples
Glucose Concentrati
on (mM)
on (mM)
on (mM)
Sports
Drink 1

0.0193

0.193

9.65

Sports
Drink 2

0.00367

0.0734

3.67

Undigeste
d Sugar

0.0274

0.274

13.7

Digested
Sugar 1

-0.0028

-0.056

-2.8

Digested
Sugar 2

0.271

2.71

135.5

Super
Sugar A1

0.0577

0.577

28.85

Super
Sugar A2

0.123

0.615

30.75

Super
Sugar B1

0.0231

0.231

11.55

Super
Sugar B2

0.0318

0.159

7.95

TABLE 7. VALUES FOR AVERAGE GLUCOSE CONCENTRATION OF EACH ORIGINAL SOLUTION FOR EACH
SAMPLE OF UNKNOWN GLUCOSE CONCENTRATION.

Original Glucose Solution

Concentration (mM)
9.65
3.67
13.7
-2.8
135.5
28.85
30.75
11.55
7.95

Unknown Samples
Sports Drink 1
Sports Drink 2
Undigested Sugar
Digested Sugar 1
Digested Sugar 2
Super Sugar A1
Super Sugar A2
Super Sugar B1
Super Sugar B2

Average Glucose
concentration (mM)
6.66
13.7
66.35
29.8
9.75

4B DATA:

Standard Curve of Retention Volume vs. Log of Apparent Molecular Weight

f(x) = - 8.62x + 54.55
R = 1

FIGURE 2. STANDARD CURVE OF RETENTION VOLUME VS THE LOG OF APPARENT MOLECULAR

WEIGHT OF STANDARDS THAT INCLUDED DNP-GLYCINE, HEMOGLOBIN, AND BLUE DEXTRAN-2000.

TABLE 8. DATA COLLECTED OF RETENTION VOLUMES AND MOLECULAR WEIGHTS OF EACH OF THE
STANDARDS.

Samples

Retention
Volume (mL)
7.36

Molecular
Weight
(Da)
2,000,000

Apparent
Molecular
Weight (Da)
300,000

Color
Observation
s
Blue

Blue-Dextran
2000
Hemoglobin

13.09

64,500

64,500

Pale Red

DNP-Glycine

22.68

241

5,000

Yellow

Glucose Oxidase Molecular Weight

V R =10.56 mL
Y =8.6177 x +54.551
10.56 mL=8.6177 x +54.551

x=5.10 Da log 10 ( Apparent MW )

105.10 Da=127,270 Da

TABLE 9. VALUE CALCULATED USING THE EQUATION FROM FIGURE 4 TO DETERMINE THE MOLECULAR WEIGHT
OF GLUCOSE OXIDASE BASED ON THE RETENTION VOLUME RECORDED FROM THE EXPERIMENT.

Sample
Glucose Oxidase

Retention Volume (mL)

10.56

Molecular Weight (Da)

127,270

Percent Error:
Literature Molecular Weight of Glucose Oxidase: 160 kDa or 160,000 Da (2)

|160,000 Da127270 Da|

160,000 Da

x 100=20.46 error

DISCUSSION:

The first part of the experiment, all three of the objectives were met. The amount of
glucose in a sports drink was calculated using the biosensor coupled reaction and the
consequent absorbance of ferricyanide was found, comparing the value to a standard
curve obtained from five known concentrations of glucose. The standard curve had an R2
value of 0.99756 meaning that the data points fitted the line extremely well. This also
meant therefore that the consequent absorbance valuesobtained from the unknown
samples would be very accurate in determining the concentration of glucose. The type of
specificity of glucose oxidase was also determined. Although it was found in literature that
the enzyme is stereospecific, this was evidenced by the undigested sugar concentration
value obtained from the absorbance.The undigested sugar, containing disaccharides
instead of the -D-glucose monosaccharide, had an extremely low concentration as
opposed to the digested sugar. The purpose of digesting the sugar in acid is so that the
disaccharide complex of glucose and fructose could be broken making the glucose more
free to bind to the Glucose Oxidase enzyme resulting in a higher glucose concentration.
The concentrations that were found from the two digested sugar solutions were very

10

different when they should not have been as different. Digested sugar solution 1 produced
an unexpectedly very low value which was alarming because with the sugar being
digested more glucose should be available to bind with the GOx enzyme increasing
absorbance and concentration. One possible explanation for this unexpected value is
human error, being it possible that the solution was missing the enzyme mix, in order to
catalyze both reactions so that the yellow color could form and absorbance could be
accurately measured thus giving an accurate measure for the concentration of glucose.
While the digested sugar solution 2 produced a reasonably high absorbance and
concentrated as expected because more of glucose would be exposed in the digested
form than glucose in the normal form.
This supported that the enzyme only worked when the right stereoisomer was
present as compared to the digested sugar that has nearly five times the amount of
glucose. Moreover this was evidenced by the low concentration of glucose compared to
the 7g listed on the label. The sugar in the drink we assumed to be glucose, however
different disaccharides must also have been present in order to make up the remainder of
the sugar content. Based on the analysis conducted, it appears that GOx is not absolutely
specific to glucose but rather specific to sugars that are similar in structure or
characteristics to glucose. This was determined by the analysis done on the two types of
super sugar solutions, Super Sugar A and Super Sugar B solutions. The Super Sugar B
solution was determined to be the super sugar solution that did not have added glucose
because it gave a lower absorbance value during data collection than the Super Sugar A
solution. Though it gave a lower value, it still gave a value, which means that there was
some absorbance of some glucose or glucose-like molecule. This hints to the specificity of
the Glucose Oxidase enzyme because based on the ingredients list, there is 7g of sugar in
the sports drink but the absorbance reading is so low that it is possible that the sugar in
the sports drink is not glucose. It is likely a sweetened substitute because as
demonstrated by the standards and the Super Sugar A data, glucose has a strong affinity
for GOx. That along with the fact that it is a low calorie drink, leads to the conclusion that
the sports drink did not have glucose in it or had very low amounts of glucose which likely
lead to the inability of the sugar to sustain the reaction with Glucose Oxidase and produce
a higher absorbance reading and subsequent concentration. The main sweetener in the
sports drink was sucralose.
Furthermore, this was determined by the analysis conducted on the two types of
super sugar solutions, Super Sugar A and Super Sugar B solutions. The Super Sugar B
solution was determined to be the super sugar solution that did not have added glucose
because it gave a lower absorbance value during data collection than the Super Sugar A
solution. Though it gave a lower value, it still gave a value, which means that there was
some absorbance of some glucose or glucose-like molecule. This hints to the specificity of
the Glucose Oxidase enzyme. It is not absolutely specific to glucose but rather
stereospecific to -D-glucose molecules. This can be explained, as glucose is the optimum
substrate for GOx. But GOx will bind to other sugars similar to glucose that will cause it to
get an absorbance reading when glucose is not present in the solution. This absorbance
reading is likely low due to the inability of the glucose-like sugars inability to sustain the
reaction between Glucose Oxidase because the affinity for it is not as strong as the affinity
for glucose.

11

The final objective that was achieved was to determine which super sugar solution
contained the added glucose. It was evidenced by the much greater concentration of
glucose in both dilutions that the super sugar sample A contained the added sugar. The
super sugar sample A contained 0.45175 mM as opposed to only 0.13725 mM witnessed
in the super sugar sample B. The only piece of data that appeared to be inconsistent was
the digested sugar 1 value. This may have been down to poor technique in pipet transfer,
or more likely not adding the enzyme solution, which would account for having such a low
absorbance since the second reaction did not occur.
The second experiment also went very well, particularly regarding the percent error
for the molecular weight of glucose oxidase. Using molecular sieve chromatography and
comparing the glucose oxidase retention volume to the calibration curve of the three
known molecular weights achieved the primary objective of determining the molecular
weight of the glucose oxidase. The calibration curve had a perfect R2 value of 1, which
meant that the points fitted the line perfectly. This gave a lot of confidence to finding an
accurate glucose oxidase molecular weight once the retention value was obtained. Just a
20.46% error was obtained when comparing the experimental molecular weight to the
literature value, which although seems large, was relatively low compared to previous
similar experiments.
Another, perhaps more accurate way the molecular weight may have been
calculated would have been the SDS-PAGE method. SDS-PAGE stands for sodium-dodecyl
sulfate polyacrylamide gel electrophoresis that uses electrophoretic mobility to determine
the molecular weight of proteins. SDS is a detergent that is negatively charged that binds
to proteins at a constant ratio and denatures them in order to manipulate electrophoretic
mobility so that it is solely dependent on molecular weight. This method uses markers of
known molecular weight as a reference in order to compare unknown samples to for
estimation of molecular weight once samples have been run through polyacrylamide gel.
(Kresge & Simoni, 2006) This method is a direct measure of the molecular weight as
opposed to the indirect method that was used in this experiment.
With respect to the standard dyes, a compound with a molecular weight of 40,000
Da would elute the column between DNP-Glycine and Hemoglobin. This is because the
lower limit of the fractionation range for the Sephadex G-150 gel is 5000 Da so any
standard that has a molecular weight that is at or below 5000 Da (DNP-Glycine= 241 Da)
is considered 5000 Da because there is no way to specifically determine its molecular
weight because it is out of the range of the Sephadex gel G-150. The molecular weight for
Hemoglobin is 64,500 Da, which is greater than the suggested molecular weight of 40,000
Da for the compound. That confirms the range in which the compound with a molecular
weight of 40,000 Da would fall in between DNP-Glycine and Hemoglobin. This would differ
if Sephadex G-50 gel was used that has a fractionation range of 1,500 Da -30,000 Da
because the compound would not fall within the fractionation range but would instead
exceed the upper limit making it impossible to specifically determine the molecular weight
of the compound using the Sephadex G-50 gel.
CONCLUSION:

12

To conclude, it was determined that the sports drink contained very little -Dglucose monosaccharides and instead either consisted of a different monosaccharide or
various disaccharides in order to make up the sugar content of the drink. It was identified
that super sugar A had the added sugar, and digested sugar contained more -D-glucose
than the undigested sugar sample.
With regards to molecular weight, glucose oxidase contained only 20.46%
experimental error meaning that although the method was rather inaccurate, in relative
terms the percent error was reflective of a successful attempt to determine the molecular
weight of glucose oxidase. The way the error could be reduced even further is to perhaps
use smaller graduations in order to pinpoint the retention volume to a more precise
volume rather than in 1 mL increments. Using a graduated cylinder with more graduations
therefore may have also helped yield a more accurate retention volume. Had there been
the time, it would have been a good idea to repeat the experiments and create another
two trials for glucose oxidase in order to have again a more accurate retention volume. It
was very hard to distinguish the most vibrant color for the glucose oxidase, so it would be
interesting to see, had there been more time, if a spectrophotometer could have detected
which of the test tubes had the most vibrant color.
It was learned how to experimentally determine an analyte using a biosensor and
an experimental method of determining molecular weight of a compound.
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13

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