82

Experimental Section

The Peroxidase-Glucose Oxidase Enzyme System in

the Undergraduate Laboratory

ELISA WOOLRIDGE,* SANDRA L TURCHI* and

JOHN R EDWARDS**

* Department of Chemistry

Roddy Science Center

Millersville University

Millersville, PA 17551, USA

** Department of Chemistry

Villanova University

Villanova, PA 19085, USA

stoichiometry of the overall reaction involves a two-electron

transfer from glucose to the o-dianisidine. 3 The amount of the

oxidized o-dianisidine formed is a direct measure of the amount

of glucose reacted and can, therefore, be measured quanti-

tatively.

Materials and Methods

Peroxidase-glucose oxidase (PGO) capsules were purchased

from Sigma Chemical Company (St Louis, Missouri, USA). All

other reagents were reagent grade and were purchased from

Fisher Scientific Co. A Bausch and Lomb

Spectronic 20

was used

for the colorimetric analyses. The gel filtration medium (Bio-Gel

A 0.5m) was purchased from Bio-Rad (Richmond, CA).

Standard solutions of glucose (10 ml per student) should be

prepared a day prior to use to allow for mutarotation since only

the [3-anomer is oxidized.

Introduction

A widely accepted procedure used for the colorimetric deter-

mination of glucose serves as the basis of a series of enzymatic

experiments. Both the glucose oxidase and the horseradish

peroxidase are stable enzymes and are nearly student-proof. The

enzymes are available from several sources and need not be

prepared from an organism. This feature makes this series of

experiments a nice way to introduce enzymology into the

laboratory sequence. This experiment gives the student experi-

ence with enzymes before they attempt to isolate and purify

them. The experiments can augment lectures on carbohydrates

and enzymes which usually occur early in the semester. In the

quantitative determination of glucose, the enzymes show a

tightly-coupled, stoichiometric reaction. The experiments

demonstrate several basic enzyme properties and the chromato-

graphic separation of the two enzyme activities.

Baekground

The peroxidase-glucose oxidase (PGO) enzyme system involves

a coupled reaction with glucose oxidase and peroxidase. The

substrates of the glucose oxidase are glucose and 02 and the

product is

H202

which serves as the substrate for the peroxidase.

The coupled enzyme preparation is sold as a mixture and is

usually used to measure the amount of glucose in biological

fluids.~ Other sugars and reducing compounds are not acted

upon and, therefore, do not give false "reducing sugar' values (cf

the Nelson-Somogyi assay). An identical system can be used to

measure galactose using galactose oxidase and peroxidase.

Glucose oxidase 2 is a flavin-dependent enzyme which

specifically oxidizes [3-D-glucopyranose in a two-step reaction:

Enzyme.FAD + 13-D-glucose--~

8-D-gluconolactone + Enzyme.FADH2

Enzyme.FADH_, + Oe ~ Enzyme.FAD +

H202

The 8-o-gluconolactone spontaneously hydrolyzes to yield the

l~-gluconate. Glucose oxidase has a molecular weight of about

160 0(10 dalton consisting of two identical polypeptide chain

subunits covalently linked by disulfide bonds. The subunits

contain one mole each of iron and FAD.

ttorseradish peroxidase is a heme-linked oxidase with a

molecular weight of 40 0(10 dalton that catalyzes the oxidation of

various substrates with hydrogen peroxidase. 2

11202 + o-dianisidine (colourless) P

..... idas,~,

H2O + o-dianisidine (brown)

During the reaction the oxidation state of the heme iron

undergoes a reversible Fe(ll)-Fe(llI) valence change. The

peroxidase initially reacts with the hydrogen peroxide and

becomes oxidized giving rise to complex I. This complex then

oxidizes the o-dianisidine in two successive one-electron trans-

fers which restores the enzyme to the native state. The

Experimental Protocol

The experiment is designed for two students to work together.

Care should be taken with the o-dianisidine since it might be

carcinogenic. One may substitute azino-di(ethylbenzthiazoline

sulfonate) for o-dianisidine for it is not a known carcinogen.

Period 1: Preparation of Glucose Standard Curve

Dilutions of a

stock glucose solution (100 ixg/ml) are mixed with o-dianisidine

(2.5 mg/ml, 0.1 ml) and the PGO solution (5.0 ml) and incubated

at 25°C. After 30 rain, HCI (2 drops, 2 M) is added to stop the

reaction. Each ml of the standard glucose solution contains 0.55

Ixmole of glucose. The absorption spectra of the oxidized o-

dianisidine is determined by measuring the absorbance of one

sample (use either the 80 or 100 Ixg glucose tube) at various

wavelengths. The values should be determined at least at 5 nm

intervals. Absorbance

versus

wavelength should be plotted.

The standard curve of absorbance

versus

glucose concen-

tration could be co-plotted using Ixg and ixmole on the X-axis.

This allows one to express data on either a weight or a molar

basis.

The completeness of the reaction should be determined by

observing in the colorimeter the increase in absorbance every 5

rain. If the reaction is not complete within 3(I rain, then one may

increase the incubation temperature to 30°C, increase the time of

incubation or increase the amount of enzyme added. For most of

these reactions completion of reaction is not necessary. The

progress of the oxidation of the chromogen by the peroxidase

should be plotted: ie, absorbance versus time.

Period 2a: Determination of K,,

Glucose stock solutions (20-100

Ixg/ml) are prepared and mixed with the PGO/o-dianisidine

mixture (5.l ml). The absorbance is monitored over an 8 rain

period noting the absorbance every 30 seconds. The amount of

glucose oxidized (in ixmoles) at each time interval is plotted

versus

time. The slope of each curve is determined to obtain the

initial velocity for each glucose concentration. The initial

velocity (p.mol glucose oxidized per rain) is plotted versus the

glucose concentration. A reciprocal plot (Lineweaver-Burk)

should also be prepared and the Vm~,× and Km for the reaction

determined. 4,5

Period 2b: Effects of Various Sugars on Km

The possible

inhibition of the PGO should be determined for one of the

following sugars: methyl ~x-glucoside,

myo-inositol,

glucos-

amine, sorbitol, xylose, fructose, arabinose, or mannose (2.5

mg/ml). The sugar (0.1 ml) should be added to each tube along

with the glucose and the reaction carried out in the same

manner. The initial velocity should be determined, the data co-

plotted with that in 2a and the apparent Kn,, V,,~,x and K,

determined.

Period 2c: Specificit)' of the Glucose Oxidase

Each sugar solution

mentioned in 2b should be prepared to yield concentrations of

BIOCHEMICAL EDUCATION 14(2) 1986