Enzymatic assay of 3-mercaptopyruvate sulfurtransferase activity in human red blood cells using pyruvate oxidase.
A highly sensitive method has been developed for the determination of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) activity in human red blood cells which is based on the colorimetric method for the determination of pyruvate using the coupled color enzymatic reaction with pyruvate oxidase (EC 1.2.3.3) and peroxidase (EC 1.11.1.7). The absorbance increase of the coupled color of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine and 4-aminoantipyrine at 555 nm is proportional to sulfurtransferase activity. The present method is more than 10-fold more sensitive and more reproducible than the usual methods, and does not require centrifugation, filtration, and neutralization steps. The calibration curve is linear up to 450 units/liter and the precision per run is 1.06% for 77.5 units/10(10) red blood cells (n = 10). Comparison with the lactate dehydrogenase method gave good correlation (r = 0.995). The normal value for the human red blood cell enzyme, determined with the present method on 30 healthy persons, was 63.8 +/- 14.4 units/10(10) cells (mean +/- 2SD).
Nawata
M
M
Department of Analytical Chemistry, Meiji College of Pharmacy, Tokyo, Japan.
Ogasawara
Y
Y
Kawanabe
K
K
Tanabe
S
S
D003124:Colorimetry / Q000379:methods; D004229:Dithiothreitol / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D011771:Pyruvate Oxidase / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013466:Sulfurtransferases / Q000378:metabolism*
D011773:Pyruvates; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D009195:Peroxidase; D011771:Pyruvate Oxidase; D013466:Sulfurtransferases; C021550:3-mercaptopyruvate sulphurtransferase; D005033:Ethylmaleimide; D004229:Dithiothreitol