Enantiospecificity of covalent adduct formation by benzo[a]pyrene anti-diol epoxide with human serum albumin.

Human serum albumin was reacted with the (+)- and (-)-enantiomers of r-7,t-8-dihydroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to determine if the chiral nature of the protein influences adduct formation. The alkylated proteins were analyzed directly by fluorescence line narrowing spectroscopy, and their spectra were compared to those of the model synthetic adducts N tau-(7,8,9-trihydroxy-r-7,t-8,t-9,c-10-tetrahydrobenzo[a]pyren-10- yl)histidine and 7,8,9-trihydroxy-r-7,t-8,t-9,c-10-tetrahydrobenzo[a]pyren- 10-yl N-t-BOC-alaninate ester. The results from these analyses indicated that different adducts were formed by the enantiomers of the diol epoxide. The adducted proteins were also enzymatically digested, and the 8,9-cis-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-containing adducts and hydrolysis products were isolated by boronate affinity chromatography. Diode array UV, fast atom bombardment, and on-line atmospheric pressure ionization-mass spectral analysis of the HPLC purified products indicated that the more mutagenic and tumorigenic (+)-enantiomer forms carboxylic ester adducts with the protein at either Asp(187) or Glu(188), while the (-)-enantiomer forms N tau-histidine adducts at His(146). This previously unrealized enantiospecificity of the reaction of benzo[a]pyrene anti-diol epoxide with human serum albumin has important consequences for the application of the adducts as biomarkers of internal exposure.