Purification of yeast alpha-isopropylmalate isomerase. High ionic strength hydrophobic chromatography

Abstract

alpha-Isopropylmalate isomerase, the second enzyme specific for leucine biosynthesis, can be purified from extracts of yeast utilizing a chromatographic procedure that allows separation of proteins in the presence of high concentrations of (NH4)2SO4. The purification procedure utilizes the stabilizing effect of glycerol and (NH4)2SO4 on the isomerase and their opposing effects on protein retention on valine-Sepharose and leucine-Sepharose. The method effectively separates the isomerase from fumarase, a stable internal marker protein that was co-purified in early steps. High ionic strength hydrophobic chromatography, based on differential retention as a function of the length of the hydrophobic sidearm and ionic strength, yields approximately 200-fold purified alpha-isopropylmalate isomerase and may be of general utility in purifying unstable enzymes requiring high ionic strength.