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Biol 211, Cell Biology, Fall 2008

Exercise #3: Kinetic Analysis of Glucose Oxidase

Investigator: Casey Snyder

Purpose of the Experiment

The scientific objective of this laboratory exercise is to determine the substrate specificity of glucose oxidase by performing kinetic analyses in the presence of glucose and xylose, two hexoses.

Materials

A series of 7 solutions of different concentrations of glucose
A series of 7 solutions of different concentrations of xylose
A bottle containing Assay Cocktail A
A bottle containing Assay Cocktail B
Blue automatic Pipettor (100 - 1000 ul)
Pipet tips
A Genesys 20 spectrophotometer

Procedure. Part 1. Kinetic Analysis of GO with Glucose as the Substrate

1. Obtain 22 disposable cuvettes. Set one aside as the blank. Arrange the 21 remaining cuvettes in two groups of 7.

Prepare the blank by adding the following substances to the blank cuvette: 0.9 ml assay cocktail A, 0.1 ml d H2O. Invert the tube 2X to mix.

3. Prepare the other 21 cuvettes by adding 0.9 ml of assay cocktail A to each tube.

Perform the following assay on each of the 7 glucose solutions in duplicate.

a. Balance the spectrophotometer with the blank. This will correct for any nonspecific background absorption of the 725 nm light.

b. Perform the following steps rapidly and carefully.

1. With a pipettor, add 0.1 ml of the desired glucose solution to one of the cuvettes.

2. Immediately invert the tube 2X to mix. Start timing at the second inversion.

3. Place the cuvette in the spectrophotometer and close the cuvette holder.

4. Record the Absorbance (A725) at 5-second and 30 seconds after the start of the reaction.

Procedure. Part 2. Kinetic Analysis of GO with Xylose as the Substrate

1. Obtain 22 disposable cuvettes. Set one aside as the blank. Arrange the 21 remaining cuvettes in two groups of 7.

Prepare the blank by adding the following substances to the blank cuvette: 0.9 ml assay cocktail B, 0.1 ml d H2O. Invert the tube 2X to mix.

3. Prepare the other 21 cuvettes by adding 0.9 ml of assay cocktail B to each tube.

Perform the following assay on each of the 7 xylose solutions in duplicate.

a. Balance the spectrophotometer with the blank. This will correct for any nonspecific background absorption of the 725 nm light.

b. Perform the following steps rapidly and carefully.

1. With a pipettor, add 0.1 ml of the desired xylose solution to one of the cuvettes.

2. Immediately invert the tube 2X to mix. Start timing at the second inversion.

3. Place the cuvette in the spectrophotometer and close the cuvette holder.

4. Record the Absorbance (A725) at 5-second and 30 seconds after the start of the reaction.

Results

**Table 1. Kinetic Analysis of GO with Glucose as the Substrate. Raw Absorbance Data**
Trial #	Sample	[S]mM	5 sec	30 sec
Trial 1	glu_A	20	0.054	0.228
Trial 2	glu_A	20	0.043	0.224
Trial 3	glu_A	20	0.051	0.216
Trial 1	glu_B	30	0.055	0.261
Trial 2	glu_B	30	0.052	0.292
Trial 3	glu_B	30	0.051	0.265
Trial 1	glu_C	40	0.053	0.319
Trial 2	glu_C	40	0.053	0.331
Trial 3	glu_C	40	0.049	0.331
Trial 1	glu_D	50	0.055	0.344
Trial 2	glu_D	50	0.061	0.387
Trial 3	glu_D	50	0.057	0.35
Trial 1	glu_E	100	0.081	0.477
Trial 2	glu_E	100	0.085	0.48
Trial 3	glu_E	100	0.076	0.467
Trial 1	glu_F	150	0.548	1.008
Trial 2	glu_F	150	0.507	0.971
Trial 3	glu_F	150	0.518	0.968
Trial 1	glu_G	200	0.128	0.587
Trial 2	glu_G	200	0.147	0.617
Trial 3	glu_G	200	0.166	0.578

Results

**Table 2. Kinetic Analysis of GO with Xylose as the Substrate. Raw Absorbance Data**
Trial #	Sample	[S]mM	5 sec	30 sec
Trial 1	xyl_A	20	0.003	0.01
Trial 2	xyl_A	20	0.008	0.015
Trial 3	xyl_A	20	0.007	0.015
Trial 1	xyl_B	30	0.004	0.029
Trial 2	xyl_B	30	0.003	0.028
Trial 3	xyl_B	30	0.004	0.032
Trial 1	xyl_C	40	0.029	0.043
Trial 2	xyl_C	40	0.028	0.041
Trial 3	xyl_C	40	0.033	0.049
Trial 1	xyl_D	50	0.01	0.053
Trial 2	xyl_D	50	0.005	0.049
Trial 3	xyl_D	50	0.008	0.053
Trial 1	xyl_E	100	1.21	1.64
Trial 2	xyl_E	100	1.03	1.53
Trial 3	xyl_E	100	1.3	2.08
Trial 1	xyl_F	150	0.039	0.188
Trial 2	xyl_F	150	0.029	0.185
Trial 3	xyl_F	150	0.048	0.225
Trial 1	xyl_G	200	0.042	0.189
Trial 2	xyl_G	200	0.039	0.185
Trial 3	xyl_G	200	0.039	0.182

**Table 3. Kinetic Analysis of GO with Glucose as the Substrate. Calculation of Vi and Vi/S**
Trial #	Sample	[S]mM	Vi	Vi/[S]
Trial 1	glu_A	20	0.007	0.00035
Trial 2	glu_A	20	0.007	0.00035
Trial 3	glu_A	20	0.007	0.00035
Trial 1	glu_B	30	0.008	0.000266666666666667
Trial 2	glu_B	30	0.01	0.000333333333333333
Trial 3	glu_B	30	0.009	0.0003
Trial 1	glu_C	40	0.011	0.000275
Trial 2	glu_C	40	0.011	0.000275
Trial 3	glu_C	40	0.011	0.000275
Trial 1	glu_D	50	0.012	0.00024
Trial 2	glu_D	50	0.013	0.00026
Trial 3	glu_D	50	0.012	0.00024
Trial 1	glu_E	100	0.016	0.00016
Trial 2	glu_E	100	0.016	0.00016
Trial 3	glu_E	100	0.016	0.00016
Trial 1	glu_F	150	0.018	0.00012
Trial 2	glu_F	150	0.019	0.000126666666666667
Trial 3	glu_F	150	0.018	0.00012
Trial 1	glu_G	200	0.018	0.00009
Trial 2	glu_G	200	0.019	0.000095
Trial 3	glu_G	200	0.016	0.00008

**Table 4. Kinetic Analysis of GO with Xylose as the Substrate. Calculation of Vi and Vi/S**
Trial #	Sample	[S]mM	Vi	Vi/[S]
Trial 1	xyl_A	20	0	0
Trial 2	xyl_A	20	0	0
Trial 3	xyl_A	20	0	0
Trial 1	xyl_B	30	0.001	0.0000333333333333333
Trial 2	xyl_B	30	0.001	0.0000333333333333333
Trial 3	xyl_B	30	0.001	0.0000333333333333333
Trial 1	xyl_C	40	0.001	0.000025
Trial 2	xyl_C	40	0.001	0.000025
Trial 3	xyl_C	40	0.001	0.000025
Trial 1	xyl_D	50	0.002	0.00004
Trial 2	xyl_D	50	0.002	0.00004
Trial 3	xyl_D	50	0.002	0.00004
Trial 1	xyl_E	100	0.017	0.00017
Trial 2	xyl_E	100	0.02	0.0002
Trial 3	xyl_E	100	0.031	0.00031
Trial 1	xyl_F	150	0.006	0.00004
Trial 2	xyl_F	150	0.006	0.00004
Trial 3	xyl_F	150	0.007	0.0000466666666666667
Trial 1	xyl_G	200	0.006	0.00003
Trial 2	xyl_G	200	0.006	0.00003
Trial 3	xyl_G	200	0.006	0.00003

**Table 5. Calculation of Vi and Average 1/Vi**
Substrate	[S]mM	mean Vi	mean Vi/[S]
glu_A	20	0.007	0.00035
glu_B	30	0.009	0.0003
glu_C	40	0.011	0.000275
glu_D	50	0.0123333333333333	0.000246666666666667
glu_E	100	0.016	0.00016
glu_F	150	0.0183333333333333	0.000122222222222222
glu_G	200	0.0176666666666667	0.0000883333333333333
xyl_A	20	0	0
xyl_B	30	0.001	0.0000333333333333333
xyl_C	40	0.001	0.000025
xyl_D	50	0.002	0.00004
xyl_E	100	0.0226666666666667	0.000226666666666667
xyl_F	150	0.00633333333333333	0.0000422222222222222
xyl_G	200	0.006	0.00003

Glucose Vmax = 0.0212675159235669

Glucose Km = 40.7643312101911

Xylose Vmax = 0

Xylose Km = -200

Lessons

Link to Lesson 1 | Link to Lesson 2

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