Ion-exchange Separation and Fluorescence Measurement of Isozyme Activity in Biological Sample

Abstract

A flow injection system with a stream-switching valve for continuous monitoring of the isozyme activity of lactic dehydrogenase (EC 1.1.1.27, LDH) after liquid chromatographic separation is described.
Sample injec ted into micro column (1.5φ×20 mm) system of DEAE-sepharose and QAEsephadex, was eluted stepwise with Tris-buffered sodium chloride (15, 30, 75, 100 and 150 mmol·dm^-3). The separated isozymes were subsequently mixed with the enzyme reagen ts and immediately passed to the fluorescence detector to measure a sample background before the beginning of the enzyme reaction. After the initial measurement of background fluorescence, the mixture, which was passed to the reaction coil by the stream-switching valve, underwent a series of enzyme reactions, ultimately resulting in the formation of NADH. The fluorescence intensity of NADH was measured finally with the same detector.
Isozyme activity was determined by subtracting the area of sample background from the area of fluorescent NADH.
A major advantage of this detection system is the ability to carry out continuous monitoring of the isozyme activity and the sample background. In addition, LDH isozyme in urine containing salt s and inhibitors is purified and measured successfully by using a corridor of a hollow fiber (cuprammonium rayon) on lined to the flow injection system.
The present devic e is useful for the measurement of isozyme activity in various biological samples.