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.
Haugen
D A
DA
Biological and Medical Research Division, Argonne National Laboratory, Illinois 60439-4833.
D000611:Aminobiphenyl Compounds / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D002273:Carcinogens / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000737:chemistry*; D004912:Erythrocytes / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007460:Iodoacetamide / Q000031:analogs & derivatives; D009603:Nitroso Compounds / Q000494:pharmacology; D012459:Salicylates; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000611:Aminobiphenyl Compounds; D001713:Biphenyl Compounds; D002273:Carcinogens; D006454:Hemoglobins; D007202:Indicators and Reagents; D009603:Nitroso Compounds; D012459:Salicylates; D013438:Sulfhydryl Compounds; C072328:4-nitrosobiphenyl; C006757:4-biphenylamine; C005487:4-iodoacetamidosalicylic acid; D003545:Cysteine; D005033:Ethylmaleimide; D007460:Iodoacetamide