A simplified method for the purification of human red blood cell glyoxalase. I. Characteristics, immunoblotting, and inhibitor studies

J Protein Chem. 1993 Apr;12(2):111-9. doi: 10.1007/BF01026032.

Abstract

Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where 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 isoelectric point value of 5.1. The KM value for methylglyoxal-glutathione hemithioacetal was 192 +/- 8 microM and the kcat value was 10.9 +/- 0.2 x 10(4) min-1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had a Ki value of 0.16 +/- 0.04 microM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with a Ki value of 3.12 +/- 0.88 microM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with a Ki 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 purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies
  • Blotting, Western
  • Cells, Cultured
  • Chromatography, Affinity
  • Chromatography, Gel
  • Electrophoresis, Polyacrylamide Gel
  • Erythrocytes / enzymology*
  • Glutathione / analogs & derivatives
  • Glutathione / pharmacology
  • Humans
  • Lactoylglutathione Lyase / antagonists & inhibitors
  • Lactoylglutathione Lyase / isolation & purification*
  • Lactoylglutathione Lyase / metabolism

Substances

  • Antibodies
  • S-(4-nitrocarbobenzoxy)glutathione
  • Lactoylglutathione Lyase
  • Glutathione
  • hexylglutathione