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Biol 211, Cell Biology, Fall 2008

Exercise #3: Kinetic Analysis of Glucose Oxidase

Investigator: Andrew Buckner

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	0.045	0.218
Trial 2	glu_A	20	0.046	0.213
Trial 3	glu_A	20	0.042	0.218
Trial 1	glu_B	30	0.046	0.266
Trial 2	glu_B	30	0.059	0.286
Trial 3	glu_B	30	0.046	0.284
Trial 1	glu_C	40	0.049	0.315
Trial 2	glu_C	40	0.058	0.333
Trial 3	glu_C	40	0.068	0.338
Trial 1	glu_D	50	0.062	0.386
Trial 2	glu_D	50	0.059	0.368
Trial 3	glu_D	50	0.068	0.38
Trial 1	glu_E	100	0.088	0.484
Trial 2	glu_E	100	0.092	0.508
Trial 3	glu_E	100	0.093	0.497
Trial 1	glu_F	150	0.089	0.537
Trial 2	glu_F	150	0.111	0.565
Trial 3	glu_F	150	0.087	0.538
Trial 1	glu_G	200	0.113	0.597
Trial 2	glu_G	200	0.122	0.629
Trial 3	glu_G	200	0.114	0.606

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	0.013	0.047
Trial 2	xyl_A	20	0.011	0.034
Trial 3	xyl_A	20	0.007	0.029
Trial 1	xyl_B	30	0.009	0.04
Trial 2	xyl_B	30	0.01	0.043
Trial 3	xyl_B	30	0.012	0.044
Trial 1	xyl_C	40	0.014	0.06
Trial 2	xyl_C	40	0.018	0.061
Trial 3	xyl_C	40	0.012	0.056
Trial 1	xyl_D	50	0.014	0.072
Trial 2	xyl_D	50	0.016	0.076
Trial 3	xyl_D	50	0.027	0.085
Trial 1	xyl_E	100	0.035	0.148
Trial 2	xyl_E	100	0.038	0.15
Trial 3	xyl_E	100	0.036	0.148
Trial 1	xyl_F	150	0.048	0.216
Trial 2	xyl_F	150	0.05	0.219
Trial 3	xyl_F	150	0.062	0.232
Trial 1	xyl_G	200	0.058	0.28
Trial 2	xyl_G	200	0.061	0.278
Trial 3	xyl_G	200	0.051	0.271

**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	0.009	0.0003
Trial 2	glu_B	30	0.009	0.0003
Trial 3	glu_B	30	0.01	0.000333333333333333
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	0.013	0.00026
Trial 2	glu_D	50	0.012	0.00024
Trial 3	glu_D	50	0.012	0.00024
Trial 1	glu_E	100	0.016	0.00016
Trial 2	glu_E	100	0.017	0.00017
Trial 3	glu_E	100	0.016	0.00016
Trial 1	glu_F	150	0.018	0.00012
Trial 2	glu_F	150	0.018	0.00012
Trial 3	glu_F	150	0.018	0.00012
Trial 1	glu_G	200	0.019	0.000095
Trial 2	glu_G	200	0.02	0.0001
Trial 3	glu_G	200	0.02	0.0001

**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	0.001	0.00005
Trial 2	xyl_A	20	0.001	0.00005
Trial 3	xyl_A	20	0.001	0.00005
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	0.002	0.00005
Trial 2	xyl_C	40	0.002	0.00005
Trial 3	xyl_C	40	0.002	0.00005
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	0.005	0.00005
Trial 2	xyl_E	100	0.004	0.00004
Trial 3	xyl_E	100	0.004	0.00004
Trial 1	xyl_F	150	0.007	0.0000466666666666667
Trial 2	xyl_F	150	0.007	0.0000466666666666667
Trial 3	xyl_F	150	0.007	0.0000466666666666667
Trial 1	xyl_G	200	0.009	0.000045
Trial 2	xyl_G	200	0.009	0.000045
Trial 3	xyl_G	200	0.009	0.000045

**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	0.00933333333333333	0.000311111111111111
glu_C	40	0.011	0.000275
glu_D	50	0.0123333333333333	0.000246666666666667
glu_E	100	0.0163333333333333	0.000163333333333333
glu_F	150	0.018	0.00012
glu_G	200	0.0196666666666667	0.0000983333333333333
xyl_A	20	0.001	0.00005
xyl_B	30	0.001	0.0000333333333333333
xyl_C	40	0.002	0.00005
xyl_D	50	0.002	0.00004
xyl_E	100	0.00433333333333333	0.0000433333333333333
xyl_F	150	0.007	0.0000466666666666667
xyl_G	200	0.009	0.000045

Glucose Vmax = 0.0246158940397351

Glucose Km = 50.3311258278146

Xylose Vmax = 0.0809999999999998

Xylose Km = 1600

Lessons

Link to Lesson 1 | Link to Lesson 2

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