A simplified method for the purification of human red blood cell glyoxalase. I. 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 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.
Allen
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RE
Department of Chemistry and Biological Chemistry, University of Essex, Wivenhoe Park, Colchester, United Kingdom.
Lo
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Thornalley
P J
PJ
D000906:Antibodies; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007791:Lactoylglutathione Lyase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D000906:Antibodies; C045594:S-(4-nitrocarbobenzoxy)glutathione; D007791:Lactoylglutathione Lyase; D005978:Glutathione; C033550:hexylglutathione