Assay of glucose using glucose oxidase

The flavoprotein enzyme, glucose oxidase (EC 1.1.3.4), which may be obtained from Aspergillus niger, catalyses the oxidation of /3-n-glucose by atmospheric oxygen to produce D-gluconolactone, which is converted to gluconic acid with the production of hydrogen peroxide:

/3-D-glucose + 02 + H20 —» D-gluconic acid + H202

The oxidation of a-D-glucose occurs at less than 1 % of the rate of oxidation of the f3 anomer. Because these two forms exist in solution in equilibrium in the proportion of 36% (a) and 64% (/3), mutarotation of the a to the /3 form must be allowed to reach equilibrium in the sample and standards for consistent results. The inclusion of aldose-1-epimerase (glucomutarotase) (EC 5.1.3.3) in the glucose oxidase reagent will permit rapid restoration of the a-/3 equilibrium, effectively enabling the reaction to go to completion.

The rate of oxidation of other monosaccharides (e.g. galactose, mannose, xylose, arabinose and fructose) by glucose oxidase has been shown to be negligible or zero but some derivatives of glucose do react slightly, e.g. 2-deoxy-d-glucose shows a reaction rate of less than 5% of that with /3-d-glucose.

Quantitation of glucose using glucose oxidase is achieved by measurement of either the hydrogen peroxide formed or the oxygen consumed during the reaction, both of which are proportional to the /3-d-glucose content of the sample.

Measurement of the hydrogen peroxide formed

Spectrophotometric methods

The methods that were originally developed for routine use were colorimetric procedures in which the hydrogen peroxide formed was measured by monitoring the change in colour of a chromogenic oxygen acceptor in the presence of the enzyme peroxidase (EC 1.11.1.7). Such chromogens are colourless in their reduced form but exhibit characteristic colours when oxidized, and o-tolidine and o-dianisidine were among the substances originally used. However, owing to their potential carcinogenic nature, they have been superseded by other, less toxic, chemicals displaying similar chromogenic properties and offering the methodological advantages of faster reaction time and the production of a more stable coloured product.

While glucose oxidase is highly specific for /3-d-glucose, the colorimetric determination of hydrogen peroxide is far less specific and significant errors may be introduced in this second stage of the assay reaction. Difficulties will arise if the glucose oxidase preparation is contaminated with catalase, which destroys the hydrogen peroxide by converting it to oxygen and water. In addition some substances, such as ascorbic acid, glutathione and haemoglobin, interfere with the reaction by competing with the chromogen as hydrogen donors. However, some of the more recently introduced chromogens are said to minimize these effects and the reaction involving the peroxidase-catalysed oxidative coupling of 4-amino-phenazone and phenol to produce a coloured complex is widely used. Another commonly used chromogen is 2,2'-azino-di-(3-ethyl-benzthiazolone sulphonic acid), which provides a simple and sensitive assay method.

Procedure 9.1: Quantitation of glucose using glucose oxidase

Reagents

Glucose oxidase reagent

Glucose oxidase (EC 1.1.3.4) 3000 units (50 /ukaial) Peroxidase (EC 1.11.1.7) 5000 units (85 ¿ikatal) 2.2'-Azino-di-(3-ethyl-benzthiazolonej sulphonic acid (ABTS) 1.0 g Phosphate buffer (0.1 mol 1 ') pH 7.0. 1 litre

> Oxidized chromogens - see Section 2.1.1.

Method

To 4.0 ml glucose oxidase reagent add 0.1 ml sample. Mix and allow to react at 30°C for exactly 30 min. Measure Ihe absorbance at 560 nm.

Standard

A series of standard solutions of glucose (0-20 mmol 1 1) should be treated in exactly the same manner as the sample and a calibration graph drawn using the results.

Calculation

The concentration of glucose in the sample is read off the calibration graph using the absorbance value obtained for the sample.

> Enzyme electrodes -see Section 8.7.

> Biosensors - see Section 4.5.

> Oxygen electrode see Section 4.4.3.

There are numerous commercially produced kits and dry reagent test devices which are available for the determination of glucose using glucose oxidase. They contain all the required reagents although their composition may vary between manufacturers, especially with respect to the chromogenic oxygen acceptor that is used.

Electrochemical methods

The electrochemical measurement of the hydrogen peroxide produced forms the basis of instruments often referred to as glucose analysers. Several are commercially available and although the design varies from one manufacturer to another, a common feature of those that amperometrie ally measure the hydrogen peroxide produced is the use of glucose oxidase in an immobilized form. This is often incorporated in an enzyme electrode which is surrounded by a small chamber of buffered reagents into which the sample is introduced. Other similar biosensor devices have more recently been developed which demonstrate improved specificity and linear range.

Alternatively, the immobilized enzyme may be packed in a bed permitting continuous sample analysis, for example of a process stream. The sample is passed through the bed of immobilized enzyme and the hydrogen peroxide produced is monitored amperometrically. Dual channels permit simultaneous analysis of glucose and lactose or glucose and sucrose (Figure 9.21), the dis-accharides being hydrolysed enzymically and the glucose content measured. Thus in the analysis of sucrose, an immobilized sucrase breaks the glycosidic linkage to yield glucose and fructose and an immobilized mutarotase ensures the conversion of a-n-glucose to /3-D-glucose for its measurement by the glucose oxidase method. The measurement of lactose is achieved similarly using /3-galactosidase as the hydrolytic enzyme.

Measurement of oxygen consumed

Alternatively, the initial oxidation of glucose can be monitored and this is most easily achieved by measuring the amount of oxygen consumed during the reaction. An electrochemical method using a polarographic Clark oxygen electrode has been used and the first oxygen electrode to be described for the measurement of glucose in 1962 contained soluble glucose oxidase held between cuprophane membranes. Recent modifications have resulted in the incorporation of a variety of forms of immobilized glucose oxidase into the electrode.

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