Abstract
We describe a new flow injection system with a stream-switching valve for continuous monitoring of the isoenzyme activities of creatine phosphokinase (EC 2.7.3.2) after liquid chromatographic separation. Serum, injected into a mini-column (15mm×2mm) of DEAE-Sepharose, was eluted stepwise with Tris-buffered sodium chloride (30, 145 and 300mM). The separated isoenzymes were subsequently mixed with the enzyme reagents and immediately passed to the fluorescence detector to measure serum background before the beginning of the enzyme reactions. After the first measurement of background fluorescence, the mixture, which was passed to the delay coil by the stream-switching valve, underwent a series of enzyme reactions, ultimately resulting in the formation of fluorescent nicotinamide adenine dinucleotide, reduced form (NADH). The fluorescence intensity of NADH was measured again with the same detector. Isoenzyme activity was determined by subtracting the area of serum background from the area of fluorescent NADH. A major advantage of this detection system is the ability to carry out continuous monitoring of the isoenzyme activities with removal of the serum background. Fluorometric detection of the separated isoenzymes permits sensitive and unambiguous detection of three isoenzymes.
