A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates.
This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20-100 microM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5'-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A412). This A412 was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01-1.0 mM hypoxanthine (HX), 0.01-1.0 mM H2O2, or H2O2 + 100 microM FeSO4. In the presence of ROI, A412 decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A412 was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A412 was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 microM, was treated with XO + 100 microM HX.(ABSTRACT TRUNCATED AT 250 WORDS)
Suzuki
Y
Y
Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0551.
Lyall
V
V
Biber
T U
TU
Ford
G D
GD
D002374:Catalase / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism*; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism; D008668:Metallothionein / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism
D005609:Free Radicals; D007042:Hypoxanthines; D009596:Nitrophenols; D013438:Sulfhydryl Compounds; D019271:Hypoxanthine; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid; D006861:Hydrogen Peroxide; D002374:Catalase; D014969:Xanthine Oxidase; D003545:Cysteine; D010100:Oxygen; C024836:4-nitrophenol