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New developments in analysis of isoenzymes separated by "high-performance" liquid chromatography.

We have developed two enzyme analyzers for use in "high-performance" liquid chromatography. In both systems two detectors are used, placed after the column effluent has been combined with assay reagent. In one system, an absorbance detector is placed before and after a post-column reaction coil. Peaks observed at one detector are subtracted from those at the other, to produce a two-point measurement of enzyme activity. The linear dynamic range was 17--1700 U/L for lactate dehydrogenase (EC 1.1.1.27). In the other system, two reaction coils were used and a single fluorescence detector was placed at the end of each coil. These coils were kept at different temperatures, and an automated switching valve diverted equal amounts of column effluent and reagent into both coils. The fluorescence readings were then subtracted to produce a differential measurement of enzyme activity. The linear dynamic range was 20--1000 U/L. We used both systems to chromatographically analyze lactate dehydrogenase isoenzymes, and could separately determine both the distribution and activity of sample isoenzymes.