Journal of Protein Chemistry

, Volume 12, Issue 2, pp 111–119

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

  • Rosamund E. Allen
  • Theodore W. C. Lo
  • Paul J. Thornalley
Article

DOI: 10.1007/BF01026032

Cite this article as:
Allen, R.E., Lo, T.W.C. & Thornalley, P.J. J Protein Chem (1993) 12: 111. 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. TheKM value for methylglyoxal-glutathione hemithioacetal was 192±8 µM and thekcat value was 10.9±0.2 × 104 min−1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had aKi value of 0.16±0.04 µM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with aKi value of 3.12±0.88 µM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with aKi 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.

Key words

Glyoxalase I methylglyoxal glutathione S-hexylglutathione affinity chromatography immunoblotting 

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Rosamund E. Allen
    • 1
  • Theodore W. C. Lo
    • 1
  • Paul J. Thornalley
    • 1
  1. 1.Department of Chemistry and Biological ChemistryUniversity of EssexWivenhoe Park, ColchesterUnited Kingdom

Personalised recommendations