In This Lab We Will Use Glucose Oxidase Coupled To Horseradish Peroxidase

The Glucose Oxidase/Peroxidase System and Enzyme Kinetics Adapted from the paper by R.C.Bateman,Jr and J. A.Evans, J.Chem.Ed.,A240

In this lab you will use glucose oxidase coupled to horseradish peroxidase. This provides a stable system and the changes are easy to follow. Thus we should easily be able to demonstrate Michaelis-Menton Kinetics.

Glucose Oxidase (EC1.1.3.4) is an FAD-dependent oxidoreductase that catalyses the following reaction:

B-D-glucose + O2 -- D-gluconolactone + H2O

To enable the reaction to be followed, the hydrogen peroxide produced is scavenged by horseradish peroxidase (HRP), which uses an electron donor to further reduce the hydrogen peroxide to water.

A dye is commonly used as the electron donor to further reduce the hydrogen peroxide to water.

H2O2 + Donor red – Donor ox + H2O

In our case the dye will be 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS). This is a stable, nontoxic, water-soluble dye substrate that turns a brilliant blue-green colour when oxidized. Two moles of ABTS are oxidized for every mole of glucose oxidized.

Because of its high specificity, this system is a commonly used component of glucose test strips. Experimental

Sugar solutions will be made up as 1 M or greater stocks. Read the labels on the solutions supplied. The ABTS/HRP stock will be prepared for you and will contain 50 mM ABTS and 25 units/mL HRP. The glucose oxidase stock will contain 0.025 mg/mL (0.5 Sigma units/mL) Obtain an accurate measure of glucose subunit concentration by measuring the absorbance of this solution at 450 nm. The molar absorbance of flavin is 1.4 x 104 M-1 cm-1. The buffer is 0.1 M sodium phosphate at pH 4.

Assay The reaction is followed in a spectrophotometer interfaced to the build-time data collection program which is used with the polarograph. The data will be analyzed using Kaliedograph. The reaction may be monitored at 725 nm, where the molar absorptivity is 1.9 x 104 M-1 cm-1, or at 414 nm, where the molar absorptivity is 3.6 x 104 M-1 cm-1.

You will require 3 mL for each assay because this is the volume of the cuvettes to be used. i.e. 1.6 mL buffer, 100 uL glucose oxidase stock, 100 uL ABTS/HRP stock 200 uL glucose

Instead of mixing these amounts each time, we will prepare a large quantity of the three components that will not be varied in that particular experiment.

Part A. Vary the concentration of glucose oxidase, using four different values. Thus the cocktail (A) should contain: 8.0 mL buffer 0.5 mL ABTS/HRP stock 1 mL 0.1 M glucose For a run, place 1.9 mL cocktail A in a cuvette, add 0.1mL glucose oxidase stock, start the collection program, mix the contents of the cuvette using the vortex mixer and insert it into the spectrophotometer. Collect the readings over several minutes. You can stop once you see that you have enough readings to clearly define the reaction rate. Using the appropriate cell pathlength and molar absorptivity, convert to the absorbance to concentration, so that the slope of the line is in moles/min. Graph the assay rate against the milligrams of enzyme in the assay. CALCULATE THE SPECIFIC ACTIVITY FOR THE ENZYME, AND COMPARE TO THE VALUE REPORTED BY THE SUPPLIER, WHICH IS OBTAINED USING A DIFFERENT ASSAY TECHNIQUE.

Part B In this experiment, we will vary the sugar used as the substrate. Cocktail B should contain 8.0 mL buffer 0.5 mL ABTS/HRP stock 0.5 mL glucose oxidase stock For a run, place 1.8 mL cocktail B in a cuvette, add 0.2 mL 0.1 M glucose, start the collection program, mix the contents of the cuvette using the vortex mixer and insert it into the spectrophotometer. Collect the readings over several minutes. You can stop once you see that you have enough readings to clearly define the reaction rate. Using the appropriate cell pathlength and molar absorptivity, convert the absorbance to concentration, so that the slope of the line is in moles/min.

Calculate the specific activity for each sugar, tabulate this, and then normalise to glucose and show the relative sugar specificities for glucose oxidase. Compare the structures of the sugars and discuss how the differences in structure between the sugars account for the differences in reactivity/enzyme recognition. Part C The glucose concentration is to be varied incrementally from 0.1 M to 0.005 M. The glucose oxidase concentration in the initial cocktail should be set so as to achieve a reaction rate between 0.5 and 1.0 absorbance units/minute with 0.1 M glucose. Look at results from previous weeks to asses this. Using such an enzyme concentration will ensure that the reaction rates are independent of the enzyme concentration and just depend on the glucose concentration being used.

Cocktail C should contain 16 mL buffer 1.0 mL ABTS/HRP stock 1.0 mL glucose oxidase stock [change to vary the concentration of glucose oxidase. Adjust the volume of buffer to compensate for larger or smaller volumes being used]

For a run, place 1.8 mL cocktail C in a cuvette, add 0.2 mL glucose of the appropriate concentration, start the collection program, mix the contents of the cuvette using the vortex mixer and insert it into the spectrophotometer. Collect the readings over several minutes. You can stop once you see that you have enough readings to clearly define the reaction rate. Using the appropriate cell pathlength and molar absorptivity, convert the absorbance to concentration, so that the slope of the line is in moles/min.

Fit your results to the Micaelis-Menton equation, and to one of its linear derivations – the Lineweaver-Burke plot.

What is the Michaelis-Menton constant for glucose oxidase? (in mM) What is its Vmax? (in umol/min/mg protein) Calculatew the turnover number for the enzyme.

Your Introduction What is the structure of the dye? What is the enzyme number for HRP? Explain how the buffer would be prepared. Write a few lines on how an enzyme works and on Michaelis-Menton Kinetics. What do you plot to demonstrate Michaelis-Menton Kinetics. What is a Lineweaver-Burke plot.