Design of a new automatic chromatography system for efficient enzyme purification equipped with a time-shared multiple activity analyzer.
A new system equipped with a computer-controlled multiple activity analyzer has been developed for the efficient purification of multiple enzymes. The system consists of the following units: conventional enzyme fractionation system with a peristaltic pump, liquid chromatographic column, fraction collector, and uv monitor; computer-operated uv-vis spectrophotometer equipped with a thermo-regulated metal block and a flow-through type silica cuvette; personal computer; dot matrix printer; cooling facility; and automatic sampling-mixing system. The whole system is operated by a newly designed time-sharing computer program for periodic and repetitive sampling of the column eluants containing multiple kinds of enzymes and of designated assay mixtures for each enzyme and for measurement of the initial velocity of spectrophotometric signals. For example, a mixture of aspartase (EC 4.3.1.1) and malate dehydrogenase (EC 1.1.1.39) and also a mixture of these two enzymes and glutamate dehydrogenase (EC 1.4.1.3 or EC 1.4.1.4) were analyzed by the above system using gel permeation chromatography, and the two or three enzyme activities were repeatedly monitored within 4 min. Based on the above results further possibilities for the application of the system for a variety of purposes are discussed.
D000818:Animals; D001220:Aspartate Ammonia-Lyase / Q000302:isolation & purification; D001322:Autoanalysis; D002417:Cattle; D002845:Chromatography / Q000295:instrumentation / Q000379:methods; D004798:Enzymes / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000302:isolation & purification; D008838:Microcomputers; D008929:Mitochondria, Heart / Q000201:enzymology; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013053:Spectrophotometry; D013552:Swine
D004798:Enzymes; D008291:Malate Dehydrogenase; D005969:Glutamate Dehydrogenase; D001220:Aspartate Ammonia-Lyase