matthew Johnson

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	0.035	0.08	0.135	0.184
2. glu_A	20	0.039	0.081	0.131	0.185
3. glu_B	30	0.035	0.085	0.141	0.199
4. glu_B	30	0.06	0.112	0.17	0.241
5. glu_C	40	0.057	0.109	0.164	0.222
6. glu_C	40	0.038	0.085	0.147	0.199
7. glu_D	50	0.041	0.089	0.145	0.205
8. glu_D	50	0.039	0.083	0.147	0.205
9. glu_E	100	0.049	0.097	0.159	0.224
10. glu_E	100	0.05	0.106	0.161	0.226
11. glu_F	150	0.042	0.095	0.152	0.205
12. glu_F	150	0.044	0.094	0.156	0.212
13. glu_G	200	0.055	0.109	0.17	0.234
14. glu_G	200	0.039	0.089	0.144	0.206

**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	0.04	0.074	0.101	0.135
2. xyl_A	20	0.07	0.127	0.172	0.214
3. xyl_B	30	0.06	0.089	0.12	0.146
4. xyl_B	30	0.047	0.086	0.125	0.155
5. xyl_C	40	0.079	0.138	0.196	0.249
6. xyl_C	40	0.064	0.106	0.144	0.183
7. xyl_D	50	0.057	0.097	0.135	0.169
8. xyl_D	50	0.051	0.088	0.125	0.157
9. xyl_E	100	0.087	0.163	0.234	0.306
10. xyl_E	100	0.089	0.161	0.238	0.308
11. xyl_F	150	0.131	0.233	0.345	0.447
12. xyl_F	150	0.129	0.236	0.355	0.461
13. xyl_G	200	0.166	0.334	0.507	0.671
14. xyl_G	200	0.169	0.311	0.458	0.593

**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	0.003	333.333
2. glu_A	20	0.05	0.003	333.333
3. glu_B	30	0.033	0.004	250
4. glu_B	30	0.033	0.004	250
5. glu_C	40	0.025	0.004	250
6. glu_C	40	0.025	0.004	250
7. glu_D	50	0.02	0.003	333.333
8. glu_D	50	0.02	0.004	250
9. glu_E	100	0.01	0.004	250
10. glu_E	100	0.01	0.004	250
11. glu_F	150	0.0067	0.004	250
12. glu_F	150	0.0067	0.004	250
13. glu_G	200	0.005	0.004	250
14. glu_G	200	0.005	0.004	250

**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	0.002	500
2. xyl_A	20	0.05	0.003	333.333
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	0.003	333.333
8. xyl_D	50	0.02	0.002	500
9. xyl_E	100	0.01	0.005	200
10. xyl_E	100	0.01	0.005	200
11. xyl_F	150	0.0067	0.007	142.857
12. xyl_F	150	0.0067	0.008	125
13. xyl_G	200	0.005	0.011	90.909
14. xyl_G	200	0.005	0.01	100

**Table 5. Calculation of Vi and Average 1/Vi**
Substrate	[S]mM	1/[S]	mean Vi	mean 1/Vi
glu_A	20	0.05	0.003	333.333
glu_B	30	0.033	0.004	250
glu_C	40	0.025	0.004	250
glu_D	50	0.02	0.004	291.667
glu_E	100	0.01	0.004	250
glu_F	150	0.0067	0.004	250
glu_G	200	0.005	0.004	250
xyl_A	20	0.05	0.003	416.667
xyl_B	30	0.033	0.003	416.667
xyl_C	40	0.025	0.004	291.667
xyl_D	50	0.02	0.003	416.667
xyl_E	100	0.01	0.005	200
xyl_F	150	0.0067	0.008	133.929
xyl_G	200	0.005	0.01	95.455

Lessons

Link to Lesson 1 | Link to Lesson 2

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