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Biol 211, Cell Biology, Fall 2008

Exercise #3: Kinetic Analysis of Glucose Oxidase

Investigator: Ramsey Hanna

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	0.037	0.205
Trial 2	glu_A	20	0.037	0.205
Trial 3	glu_A	20	0.087	0.259
Trial 1	glu_B	30	0.051	0.283
Trial 2	glu_B	30	0.055	0.288
Trial 3	glu_B	30	0.055	0.283
Trial 1	glu_C	40	0.071	0.35
Trial 2	glu_C	40	0.073	0.35
Trial 3	glu_C	40	0.071	0.345
Trial 1	glu_D	50	0.085	0.386
Trial 2	glu_D	50	0.088	0.391
Trial 3	glu_D	50	0.091	0.393
Trial 1	glu_E	100	0.085	0.499
Trial 2	glu_E	100	0.2	0.493
Trial 3	glu_E	100	0.092	0.5
Trial 1	glu_F	150	0.112	0.563
Trial 2	glu_F	150	0.097	0.554
Trial 3	glu_F	150	0.123	0.584
Trial 1	glu_G	200	0.134	0.616
Trial 2	glu_G	200	0.113	0.58
Trial 3	glu_G	200	0.123	0.592

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	0.009	0.032
Trial 2	xyl_A	20	0.003	0.019
Trial 3	xyl_A	20	0.008	0.023
Trial 1	xyl_B	30	0.011	0.036
Trial 2	xyl_B	30	0.01	0.035
Trial 3	xyl_B	30	0.011	0.036
Trial 1	xyl_C	40	0.019	0.059
Trial 2	xyl_C	40	0.024	0.062
Trial 3	xyl_C	40	0.017	0.06
Trial 1	xyl_D	50	0.017	0.056
Trial 2	xyl_D	50	0.015	0.056
Trial 3	xyl_D	50	0.017	0.059
Trial 1	xyl_E	100	0.036	0.139
Trial 2	xyl_E	100	0.035	0.143
Trial 3	xyl_E	100	0.035	0.141
Trial 1	xyl_F	150	0.065	0.289
Trial 2	xyl_F	150	0.059	0.221
Trial 3	xyl_F	150	0.066	0.226
Trial 1	xyl_G	200	0.066	0.281
Trial 2	xyl_G	200	0.059	0.248
Trial 3	xyl_G	200	0.067	0.282

**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	0.009	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	0.012	0.00024
Trial 3	glu_D	50	0.012	0.00024
Trial 1	glu_E	100	0.017	0.00017
Trial 2	glu_E	100	0.012	0.00012
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	0.019	0.000095
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	0.009	0.00006
Trial 2	xyl_F	150	0.006	0.00004
Trial 3	xyl_F	150	0.006	0.00004
Trial 1	xyl_G	200	0.009	0.000045
Trial 2	xyl_G	200	0.008	0.00004
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	0.009	0.0003
glu_C	40	0.011	0.000275
glu_D	50	0.012	0.00024
glu_E	100	0.015	0.00015
glu_F	150	0.018	0.00012
glu_G	200	0.019	0.000095
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	0.007	0.0000466666666666667
xyl_G	200	0.00866666666666667	0.0000433333333333333

Glucose Vmax = 0.0234705882352941

Glucose Km = 47.0588235294118

Xylose Vmax = 0.0585000000000001

Xylose Km = 1150

Lessons

Link to Lesson 1 | Link to Lesson 2

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