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

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.