Charge-shift strategy for isolation of hemoglobin-carcinogen adducts formed at the beta 93 cysteine sulfhydryl groups

Qualitative and quantitative analysis of human hemoglobin-carcinogen adducts has potential as a diagnostic tool for estimation of biologically effective levels of carcinogen exposure and for attaining a better understanding of individual susceptibility to chemical carcinogenesis. The purpose of this study was to devise a strategy for preanalytical enrichment of the class of covalent human hemoglobin-carcinogen adducts formed by reaction at the hemoglobin beta 93 cysteine sulfhydryl groups. The results define a charge-shift strategy in which a mixture composed of natural hemoglobin (Hb-SH) and low levels of hemoglobin-S-xenobiotic adducts (Hb-SX) is treated with an anionic sulfhydryl reagent (R-), followed by anion-exchange liquid chromatographic separation of Hb-SR- from the unreactive Hb-SX adducts. Using 4-(iodoacetamido)-salicylic acid as the charge-shift reagent, we applied the strategy to the isolation of chromatographically similar adducts with either 4-nitrosobiphenyl or [3H]-N-ethylmaleimide. The strategy was effective for adduct concentrations less than or equal to 10 mumol/mol of hemoglobin. Application of the strategy provides an adduct-enriched fraction useful for subsequent analysis using either currently available techniques or alternate chemical or biochemical techniques that may be designed to take advantage of the enrichment procedure.