Electrophoretic analysis of isoforms of glyoxalase II in clinical blood samples.
A non-denaturing polyacrylamide gel electrophoresis procedure is described for the analysis of isoforms of human red blood cell glyoxalase II (EC 3.1.2.6). A cathodic, continuous buffer electrophoresis system was developed, using a 60 g/l polyacrylamide gel with 25 g/l N,N1-methylene-bis-acrylamide in 50 mmol/l sodium succinate buffer, pH 5.5 and 1 degrees C. Staining for glyoxalase II activity involved (a) washing the gel with 50 mmol/l Tris-HCl buffer, pH 7.4 and 37 degrees C, and (b) incubation with developing agent (0.5 g/l 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide, 0.1 g/l 2,6-dichlorophenolindophenol, 1 mmol/l S-D-lactoylglutathione in 100 mmol/l Tris-HCl, pH 7.4). Two activity bands were found in all human blood samples studied to date, probably reflecting the presence of two major isoforms. Optimal sample storage and preparation procedures and validation studies are described. This method is currently in use in an ongoing investigation of glyoxalase II isoforms in disease processes.
McLellan
A C
AC
Department of Chemistry and Biological Chemistry, University of Essex.
Thornalley
P J
PJ
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes / Q000097:blood; D013194:Staining and Labeling; D013869:Thiolester Hydrolases / Q000097:blood*; D000099301:Hydroxyacylglutathione Hydrolase
D007527:Isoenzymes; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase