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Biol 211, Cell Biology, Fall 2008

Exercise #3: Kinetic Analysis of Glucose Oxidase

Investigator: Taylor Saulman

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	0.087	0.265
Trial 2	glu_A	20	0.052	0.265
Trial 3	glu_A	20	0.043	0.235
Trial 1	glu_B	30	0.062	0.303
Trial 2	glu_B	30	0.066	0.292
Trial 3	glu_B	30	0.068	0.302
Trial 1	glu_C	40	0.087	0.356
Trial 2	glu_C	40	0.089	0.368
Trial 3	glu_C	40	0.078	0.355
Trial 1	glu_D	50	0.095	0.391
Trial 2	glu_D	50	0.095	0.402
Trial 3	glu_D	50	0.096	0.396
Trial 1	glu_E	100	0.133	0.545
Trial 2	glu_E	100	0.13	0.531
Trial 3	glu_E	100	0.121	0.526
Trial 1	glu_F	150	0.145	0.602
Trial 2	glu_F	150	0.125	0.571
Trial 3	glu_F	150	0.141	0.572
Trial 1	glu_G	200	0.135	0.623
Trial 2	glu_G	200	0.152	0.581
Trial 3	glu_G	200	0.139	0.617

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	0.024	0.095
Trial 2	xyl_A	20	0.015	0.054
Trial 3	xyl_A	20	0.012	0.053
Trial 1	xyl_B	30	0.017	0.067
Trial 2	xyl_B	30	0.021	0.064
Trial 3	xyl_B	30	0.017	0.051
Trial 1	xyl_C	40	0.016	0.06
Trial 2	xyl_C	40	0.023	0.074
Trial 3	xyl_C	40	0.018	0.059
Trial 1	xyl_D	50	0.022	0.08
Trial 2	xyl_D	50	0.024	0.081
Trial 3	xyl_D	50	0.022	0.085
Trial 1	xyl_E	100	0.044	0.146
Trial 2	xyl_E	100	0.042	0.154
Trial 3	xyl_E	100	0.036	0.141
Trial 1	xyl_F	150	0.061	0.225
Trial 2	xyl_F	150	0.057	0.209
Trial 3	xyl_F	150	0.055	0.216
Trial 1	xyl_G	200	0.071	0.243
Trial 2	xyl_G	200	0.084	0.283
Trial 3	xyl_G	200	0.083	0.261

**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	0.009	0.00045
Trial 3	glu_A	20	0.008	0.0004
Trial 1	glu_B	30	0.01	0.000333333333333333
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	0.016	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	0.018	0.00012
Trial 3	glu_F	150	0.017	0.000113333333333333
Trial 1	glu_G	200	0.02	0.0001
Trial 2	glu_G	200	0.017	0.000085
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	0.003	0.00015
Trial 2	xyl_A	20	0.002	0.0001
Trial 3	xyl_A	20	0.002	0.0001
Trial 1	xyl_B	30	0.002	0.0000666666666666667
Trial 2	xyl_B	30	0.002	0.0000666666666666667
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	0.003	0.00006
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	0.006	0.00004
Trial 1	xyl_G	200	0.007	0.000035
Trial 2	xyl_G	200	0.008	0.00004
Trial 3	xyl_G	200	0.007	0.000035

**Table 5. Calculation of Vi and Average 1/Vi**
Substrate	[S]mM	mean Vi	mean Vi/[S]
glu_A	20	0.008	0.0004
glu_B	30	0.00933333333333333	0.000311111111111111
glu_C	40	0.011	0.000275
glu_D	50	0.012	0.00024
glu_E	100	0.016	0.00016
glu_F	150	0.0176666666666667	0.000117777777777778
glu_G	200	0.0186666666666667	0.0000933333333333333
xyl_A	20	0.00233333333333333	0.000116666666666667
xyl_B	30	0.00166666666666667	0.0000555555555555556
xyl_C	40	0.002	0.00005
xyl_D	50	0.00233333333333333	0.0000466666666666667
xyl_E	100	0.004	0.00004
xyl_F	150	0.00633333333333333	0.0000422222222222222
xyl_G	200	0.00733333333333333	0.0000366666666666667

Glucose Vmax = 0.0219130434782609

Glucose Km = 34.7826086956522

Xylose Vmax = 0.009625

Xylose Km = 62.5

Lessons

Link to Lesson 1 | Link to Lesson 2

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