Conversion of a hemoglobin alpha chain aspartate(47) ester to N-(2,3-dihydroxypropyl)asparagine as a method for identification of the principal binding site for benzo[a]pyrene anti-diol epoxide.

B W Day, P L Skipper, R H Rich, S Naylor, S R Tannenbaum

Index: Chem. Res. Toxicol. 4(3) , 359-63, (1991)

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Abstract

Human hemoglobin was alkylated with (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and then treated with aqueous (+/-)-3-amino-1,2-propanediol to convert alkylated carboxyl side chains to N-(2,3-dihydroxypropyl) amides. Tryptic peptides produced from the modified protein were subjected to affinity chromatography on phenylboronic acid. The bound fraction was further purified by HPLC on C-4 reverse-phase medium to yield one modified peptide, which was identified as the Thr(41)-Lys(56) peptide of the alpha chain by amino acid analysis, Edman sequencing analysis, and FAB-MS. Limited direct evidence from this study and further indirect evidence from previous work identify Asp(47) alpha as the amino acid reacting with BPDE. The only other likely sites would be the C-terminal carboxyl groups of either the alpha or beta chain. Possible reasons for the site selectivity of the alkylation of human hemoglobin by BPDE are discussed.