Purification and partial characterization of glyoxalase I from bovine brain

Glyoxalase I was purified to homogeneity from bovine brain using affinity c hromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The e nzyme was a dimer (44,000 Daltons) composed of, apparently, identical subun its (22,000 Daltons), as shown by SDS electrophoresis, and contained one mo le of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysi s against EDTA, but the activity of the apoenzyme obtained was not complete ly restored after addition of Co2+ and Zn2+ (< 25%), while a recovery of 50 % was obtained after addition of Mg2+. The enzyme was inhibited by S-bromob enzyl-glutathione and S-p-nitrobenzylglutathione with a K-i value of 21 muM and 32 muM, respectively. The highest dissociation constant observed for t he brain enzyme with respect to that reported for human erythrocytes, or ot her mammalian forms of enzyme could be related to a tissue-specific depende nce of the glyoxalase I activity.