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Biol 211, Cell Biology, Fall 2008

Exercise #3: Kinetic Analysis of Glucose Oxidase

Investigator: Aliza Sial

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	0.027	0.099
Trial 2	glu_A	20	0.023	0.105
Trial 3	glu_A	20	0.026	0.095
Trial 1	glu_B	30	0.026	0.151
Trial 2	glu_B	30	0.028	0.157
Trial 3	glu_B	30	0.017	0.097
Trial 1	glu_C	40	0.028	0.128
Trial 2	glu_C	40	0.028	0.167
Trial 3	glu_C	40	0.041	0.197
Trial 1	glu_D	50	0.034	0.2
Trial 2	glu_D	50	0.036	0.215
Trial 3	glu_D	50	0.031	0.168
Trial 1	glu_E	100	0.053	0.297
Trial 2	glu_E	100	0.051	0.3
Trial 3	glu_E	100	0.044	0.261
Trial 1	glu_F	150	0.071	0.349
Trial 2	glu_F	150	0.06	0.353
Trial 3	glu_F	150	0.076	0.425
Trial 1	glu_G	200	0.067	0.447
Trial 2	glu_G	200	0.077	0.381
Trial 3	glu_G	200	0.095	0.503

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	0.049
Trial 2	xyl_A	20	0.011	0.029
Trial 3	xyl_A	20	0.015	0.025
Trial 1	xyl_B	30	0.011	0.023
Trial 2	xyl_B	30	0.015	0.03
Trial 3	xyl_B	30	0.035	0.05
Trial 1	xyl_C	40	0.02	0.042
Trial 2	xyl_C	40	0.014	0.026
Trial 3	xyl_C	40	0.02	0.044
Trial 1	xyl_D	50	0.019	0.038
Trial 2	xyl_D	50	0.02	0.04
Trial 3	xyl_D	50	0.022	0.035
Trial 1	xyl_E	100	0.023	0.052
Trial 2	xyl_E	100	0.02	0.047
Trial 3	xyl_E	100	0.018	0.05
Trial 1	xyl_F	150	0.025	0.085
Trial 2	xyl_F	150	0.02	0.078
Trial 3	xyl_F	150	0.026	0.077
Trial 1	xyl_G	200	0.028	0.082
Trial 2	xyl_G	200	0.025	0.13
Trial 3	xyl_G	200	0.031	0.09

**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	0.003	0.00015
Trial 2	glu_A	20	0.003	0.00015
Trial 3	glu_A	20	0.003	0.00015
Trial 1	glu_B	30	0.005	0.000166666666666667
Trial 2	glu_B	30	0.005	0.000166666666666667
Trial 3	glu_B	30	0.003	0.0001
Trial 1	glu_C	40	0.004	0.0001
Trial 2	glu_C	40	0.006	0.00015
Trial 3	glu_C	40	0.006	0.00015
Trial 1	glu_D	50	0.007	0.00014
Trial 2	glu_D	50	0.007	0.00014
Trial 3	glu_D	50	0.005	0.0001
Trial 1	glu_E	100	0.01	0.0001
Trial 2	glu_E	100	0.01	0.0001
Trial 3	glu_E	100	0.009	0.00009
Trial 1	glu_F	150	0.011	0.0000733333333333333
Trial 2	glu_F	150	0.012	0.00008
Trial 3	glu_F	150	0.014	0.0000933333333333333
Trial 1	glu_G	200	0.015	0.000075
Trial 2	glu_G	200	0.012	0.00006
Trial 3	glu_G	200	0.016	0.00008

**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	0
Trial 1	xyl_B	30	0	0
Trial 2	xyl_B	30	0.001	0.0000333333333333333
Trial 3	xyl_B	30	0.001	0.0000333333333333333
Trial 1	xyl_C	40	0.001	0.000025
Trial 2	xyl_C	40	0	0
Trial 3	xyl_C	40	0.001	0.000025
Trial 1	xyl_D	50	0.001	0.00002
Trial 2	xyl_D	50	0.001	0.00002
Trial 3	xyl_D	50	0.001	0.00002
Trial 1	xyl_E	100	0.001	0.00001
Trial 2	xyl_E	100	0.001	0.00001
Trial 3	xyl_E	100	0.001	0.00001
Trial 1	xyl_F	150	0.002	0.0000133333333333333
Trial 2	xyl_F	150	0.002	0.0000133333333333333
Trial 3	xyl_F	150	0.002	0.0000133333333333333
Trial 1	xyl_G	200	0.002	0.00001
Trial 2	xyl_G	200	0.004	0.00002
Trial 3	xyl_G	200	0.002	0.00001

**Table 5. Calculation of Vi and Average 1/Vi**
Substrate	[S]mM	mean Vi	mean Vi/[S]
glu_A	20	0.003	0.00015
glu_B	30	0.00433333333333333	0.000144444444444444
glu_C	40	0.00533333333333333	0.000133333333333333
glu_D	50	0.00633333333333333	0.000126666666666667
glu_E	100	0.00966666666666666	0.0000966666666666667
glu_F	150	0.0123333333333333	0.0000822222222222222
glu_G	200	0.0143333333333333	0.0000716666666666667
xyl_A	20	0.000666666666666667	0.0000333333333333333
xyl_B	30	0.000666666666666667	0.0000222222222222222
xyl_C	40	0.000666666666666667	0.0000166666666666667
xyl_D	50	0.001	0.00002
xyl_E	100	0.001	0.00001
xyl_F	150	0.002	0.0000133333333333333
xyl_G	200	0.00266666666666667	0.0000133333333333333

Glucose Vmax = 0.0247021276595745

Glucose Km = 144.68085106383

Xylose Vmax = 0.004

Xylose Km = 100

Lessons

Link to Lesson 1 | Link to Lesson 2

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