A SIMPLIFIED METHOD FOR THE PURIFICATION OF HUMAN RED-BLOOD-CELL GLYOXALASE .1. CHARACTERISTICS, IMMUNOBLOTTING, AND INHIBITOR STUDIES

Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography wi th a modified concentration gradient of S-hexylglutathione for elution . The pure protein had a specific activity of 1830 U/mg of protein, wh ere the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelect ric point value of 5.1. The K(M) value for methylglyoxal-glutathione h emithioacetal was 192 +/- 8 muM and the k(cat) value was 10.9 +/- 0.2 x 10(4) min-1 (N=15). The glyoxalase I inhibitor S-p-bromobenzylglutat hione had a K(i) value of 0.16 +/- 0.04 muM and S-p-nitrobenzoxycarbon ylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with a K(i) value of 3.12 +/- 0.88 muM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with a K (i) value of 18 +/- 8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high pur ity with simple low pressure chromatographic techniques with a moderat e but adequate yield for small-scale preparations.