Mark Morrison

Biol 211, Cell Biology, Fall 2007

Kinetic Analysis of Glucose Oxidase

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 15 disposable cuvettes. Set one aside as the blank. Arrange the 14 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 14 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 15-second intervals for 60 seconds.

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

1. Obtain 15 disposable cuvettes. Set one aside as the blank. Arrange the 14 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 14 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 15-second intervals for 60 seconds.

Results

**Table 1. Kinetic Analysis of GO with Glucose as the Substrate. Raw Absorbance Data**
Sample	[S]mM	15 sec	30 sec	45 sec	60 sec
1. glu_A	20	0.28	0.42	0.59	0.77
2. glu_A	20	0.28	0.46	0.63	0.82
3. glu_B	30	0.37	0.53	0.75	0.96
4. glu_B	30	0.39	0.52	0.74	0.95
5. glu_C	40	0.39	0.59	0.83	1.07
6. glu_C	40	0.39	0.62	0.85	1.08
7. glu_D	50	0.41	0.68	0.92	1.24
8. glu_D	50	0.38	0.66	0.93	1.17
9. glu_E	100	0.42	0.74	1.05	1.35
10. glu_E	100	0.46	0.77	1.25	1.36
11. glu_F	150	0.44	0.78	1.12	1.4
12. glu_F	150	0.44	0.77	1.1	1.4
13. glu_G	200	0.45	0.79	1.13	1.44
14. glu_G	200	0.53	0.88	1.23	1.52

**Table 2. Kinetic Analysis of GO with Xylose as the Substrate. Raw Absorbance Data**
Sample	[S]mM	15 sec	30 sec	45 sec	60 sec
1. xyl_A	20	0.12	0.2	0.28	0.37
2. xyl_A	20	0.17	0.24	0.37	0.46
3. xyl_B	30	0.05	0.07	0.11	0.14
4. xyl_B	30	0.08	0.13	0.17	0.22
5. xyl_C	40	0.08	0.14	0.19	0.24
6. xyl_C	40	0.09	0.14	0.19	0.24
7. xyl_D	50	0.16	0.27	0.37	0.47
8. xyl_D	50	0.1	0.17	0.24	0.36
9. xyl_E	100	0.15	0.26	0.36	0.46
10. xyl_E	100	0.17	0.27	0.38	0.46
11. xyl_F	150	0.21	0.34	0.48	0.62
12. xyl_F	150	0.23	0.36	0.49	0.61
13. xyl_G	200	0.2	0.34	0.48	0.62
14. xyl_G	200	0.27	0.44	0.63	0.79

**Table 3. Kinetic Analysis of GO with Glucose as the Substrate. Calculation of Vi and 1/Vi**
Sample	[S]mM	1/[S]	Vi	1/Vi
1. glu_A	20	0.05	0.01	100
2. glu_A	20	0.05	0.012	83.333
3. glu_B	30	0.033	0.013	76.923
4. glu_B	30	0.033	0.012	83.333
5. glu_C	40	0.025	0.015	66.667
6. glu_C	40	0.025	0.015	66.667
7. glu_D	50	0.02	0.017	58.824
8. glu_D	50	0.02	0.018	55.556
9. glu_E	100	0.01	0.021	47.619
10. glu_E	100	0.01	0.026	38.462
11. glu_F	150	0.0067	0.023	43.478
12. glu_F	150	0.0067	0.022	45.455
13. glu_G	200	0.005	0.023	43.478
14. glu_G	200	0.005	0.023	43.478

**Table 4. Kinetic Analysis of GO with Xylose as the Substrate. Calculation of Vi and 1/Vi**
Sample	[S]mM	1/[S]	Vi	1/Vi
1. xyl_A	20	0.05	0.005	200
2. xyl_A	20	0.05	0.007	142.857
3. xyl_B	30	0.033	0.002	500
4. xyl_B	30	0.033	0.003	333.333
5. xyl_C	40	0.025	0.004	250
6. xyl_C	40	0.025	0.003	333.333
7. xyl_D	50	0.02	0.007	142.857
8. xyl_D	50	0.02	0.005	200
9. xyl_E	100	0.01	0.007	142.857
10. xyl_E	100	0.01	0.007	142.857
11. xyl_F	150	0.0067	0.009	111.111
12. xyl_F	150	0.0067	0.009	111.111
13. xyl_G	200	0.005	0.009	111.111
14. xyl_G	200	0.005	0.012	83.333

**Table 5. Calculation of Vi and Average 1/Vi**
Substrate	[S]mM	1/[S]	mean Vi	mean 1/Vi
glu_A	20	0.05	0.011	91.667
glu_B	30	0.033	0.013	80.128
glu_C	40	0.025	0.015	66.667
glu_D	50	0.02	0.018	57.19
glu_E	100	0.01	0.024	43.041
glu_F	150	0.0067	0.023	44.467
glu_G	200	0.005	0.023	43.478
xyl_A	20	0.05	0.006	171.429
xyl_B	30	0.033	0.003	416.667
xyl_C	40	0.025	0.004	291.667
xyl_D	50	0.02	0.006	171.429
xyl_E	100	0.01	0.007	142.857
xyl_F	150	0.0067	0.009	111.111
xyl_G	200	0.005	0.01	97.222

Lessons

Link to Lesson 1 | Link to Lesson 2

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