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Journal of Protein Chemistry

, Volume 12, Issue 2, pp 111–119 | Cite as

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

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. TheK M 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 aK i value of 0.16±0.04 µM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with aK i value of 3.12±0.88 µM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with aK 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 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 

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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

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