Info

L-Arginine

L-Orni thine/urea

Urea-selective electrode

Many methods have been developed in which a product of the reaction is chemically modified to produce a substance with a particular spectral property. The inorganic phosphate released by the hydrolysis of phosphate esters may be measured by simple chemical methods (Fiske and Subbarow) after the enzyme reaction has been stopped. Such techniques are often convenient but do not lend themselves to the measurement of initial velocity.

Coupled enzyme assays provide a good alternative to chemical modification and permit a kinetic technique to be employed. In a coupled assay, the rate at which the product is formed is measured by using the product of the reaction as a substrate for a second enzyme reaction which can be monitored more easily. Coupled assays offer great flexibility in enzyme methodology while still retaining all the advantages of continuous monitoring techniques and are illustrated by Procedure 8.5.

PROCEDURE 8.5: Spectrophotometric assay of »-amino acid oxidase (kinetic)

Test reaction — catalysed by n-amino acid oxidase

Indicator reaction — catalysed by glutamate dehydrogenase

NH, + HOOCCH:CH,COCOOir - NADH —» HOOCCH2CII2C]lNII2CC)OH + NAD

Assay reagent

Glycvlglycine buffer pH 8.3

(0.1 mol 1 ')

2.05 ml

2-Oxoglutarate

(0.2 mol I ')

0.20 ml

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