Partial Purification and Characterization of Glyoxalase I from Porcine Erythrocytes

Abstract

Glyoxalase I from porcine erythrocytes has been purified about 2000‐fold by ion‐exchange chromatography on SE‐Sephadex, CM‐cellulose and DEAE‐cellulose columns. The isoelectric point was estimated to be at about pH 5, and the molecular weight was 52000 as determined by gel filtration. The kinetics of the enzymatic reaction was studied and the Km value was determined to be 0.19 mM, which differs significantly from the Km value of the yeast glyoxalase I. The enzymatic activity is completely inhibited by the chelating agents EDTA and o‐phenanthroline, and activity is regained by addition of Mg2+, Mn2+, and Ca2+. The reactivation is time‐dependent, which indicates that a metal · enzyme complex is the catalytically active species. An apparent dissociation constant of 0.6 mM for the Mg2+· enzyme complex was determined. Notable differences between glyoxalase I from yeast and from porcine erythrocytes appear in molecular weight, kinetics, and reactivation of chelator‐inhibited enzyme. Copyright © 1972, Wiley Blackwell. All rights reserved