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Biol 211, Cell Biology, Fall 2008

Exercise #3: Kinetic Analysis of Glucose Oxidase

Investigator: Katherine Rawls

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	0.047	0.212
Trial 2	glu_A	20	0.045	0.216
Trial 3	glu_A	20	0.046	0.206
Trial 1	glu_B	30	0.049	0.263
Trial 2	glu_B	30	0.068	0.268
Trial 3	glu_B	30	0.053	0.264
Trial 1	glu_C	40	0.062	0.32
Trial 2	glu_C	40	0.065	0.323
Trial 3	glu_C	40	0.072	0.323
Trial 1	glu_D	50	0.071	0.36
Trial 2	glu_D	50	0.068	0.36
Trial 3	glu_D	50	0.073	0.364
Trial 1	glu_E	100	0.085	0.495
Trial 2	glu_E	100	0.088	0.484
Trial 3	glu_E	100	0.123	0.442
Trial 1	glu_F	150	0.099	0.543
Trial 2	glu_F	150	0.097	0.565
Trial 3	glu_F	150	0.098	0.568
Trial 1	glu_G	200	0.107	0.591
Trial 2	glu_G	200	0.104	0.594
Trial 3	glu_G	200	0.111	0.593

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	0.015	0.034
Trial 2	xyl_A	20	0.017	0.039
Trial 3	xyl_A	20	0.014	0.029
Trial 1	xyl_B	30	0.016	0.046
Trial 2	xyl_B	30	0.018	0.054
Trial 3	xyl_B	30	0.026	0.059
Trial 1	xyl_C	40	0.028	0.071
Trial 2	xyl_C	40	0.022	0.065
Trial 3	xyl_C	40	0.024	0.066
Trial 1	xyl_D	50	0.024	0.085
Trial 2	xyl_D	50	0.022	0.077
Trial 3	xyl_D	50	0.024	0.083
Trial 1	xyl_E	100	0.043	0.154
Trial 2	xyl_E	100	0.044	0.154
Trial 3	xyl_E	100	0.045	0.156
Trial 1	xyl_F	150	0.057	0.222
Trial 2	xyl_F	150	0.062	0.222
Trial 3	xyl_F	150	0.062	0.233
Trial 1	xyl_G	200	0.07	0.286
Trial 2	xyl_G	200	0.068	0.281
Trial 3	xyl_G	200	0.076	0.296

**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	0.006	0.0003
Trial 1	glu_B	30	0.009	0.0003
Trial 2	glu_B	30	0.008	0.000266666666666667
Trial 3	glu_B	30	0.008	0.000266666666666667
Trial 1	glu_C	40	0.01	0.00025
Trial 2	glu_C	40	0.01	0.00025
Trial 3	glu_C	40	0.01	0.00025
Trial 1	glu_D	50	0.012	0.00024
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	0.016	0.00016
Trial 3	glu_E	100	0.013	0.00013
Trial 1	glu_F	150	0.018	0.00012
Trial 2	glu_F	150	0.019	0.000126666666666667
Trial 3	glu_F	150	0.019	0.000126666666666667
Trial 1	glu_G	200	0.019	0.000095
Trial 2	glu_G	200	0.02	0.0001
Trial 3	glu_G	200	0.019	0.000095

**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	0.004	0.00004
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	0.006	0.00004
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	0.00666666666666667	0.000333333333333333
glu_B	30	0.00833333333333333	0.000277777777777778
glu_C	40	0.01	0.00025
glu_D	50	0.012	0.00024
glu_E	100	0.015	0.00015
glu_F	150	0.0186666666666667	0.000124444444444444
glu_G	200	0.0193333333333333	0.0000966666666666667
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	0.004	0.00004
xyl_F	150	0.00666666666666667	0.0000444444444444444
xyl_G	200	0.009	0.000045

Glucose Vmax = 0.0245070422535211

Glucose Km = 53.5211267605634

Xylose Vmax = 0.0809999999999998

Xylose Km = 1600

Lessons

Link to Lesson 1 | Link to Lesson 2

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