Gel electrophoretic quantitation of protein carbonyls derivatized with tritiated sodium borohydride

A method for the quantitation of protein carbonyls, which have been widely employed as markers of protein oxidative damage, is described. Protein carb onyls were derivatized with tritiated sodium borohydride and the tritiated proteins were separated on SDS-PAGE. Protein bands, visualized by Coomassie blue staining, were then excised and incubated in 30% H2O2 at 60 degrees C for 48 h. Tritium, incorporated into the proteins, was quantitated by Liqu id scintillation counting after gel solubilization by H2O2. This method can be applied to the measurement of carbonylation of specific proteins as it employs SDS-PAGE and has the advantage that unreacted (NaBH4)-H-3 in the la belling reaction mixture need not be removed. The present method, when comb ined with immunochemical detection of protein carbonyls, should be very use ful in the quantitation of oxidative damage to individual proteins. (C) 199 8 Academic Press.