Day B W, Skipper P L, Zaia J, Singh K, Tannenbaum S R
Department of Environmental & Occupational Health, University of Pittsburgh, Pennsylvania 15238.
Chem Res Toxicol. 1994 Nov-Dec;7(6):829-35. doi: 10.1021/tx00042a017.
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.
将人血清白蛋白与r-7,t-8-二羟基-t-9,t-10-环氧-7,8,9,10-四氢苯并[a]芘的(+)-和(-)-对映体反应，以确定蛋白质的手性性质是否会影响加合物的形成。通过荧光线窄化光谱法直接分析烷基化蛋白质，并将其光谱与模型合成加合物Nτ-(7,8,9-三羟基-r-7,t-8,t-9,c-10-四氢苯并[a]芘-10-基)组氨酸和7,8,9-三羟基-r-7,t-8,t-9,c-10-四氢苯并[a]芘-10-基N-t-BOC-丙氨酸酯的光谱进行比较。这些分析结果表明，二醇环氧化物的对映体形成了不同的加合物。还对加合蛋白质进行了酶消化，并通过硼酸酯亲和色谱法分离出含8,9-顺-二羟基-7,8,9,10-四氢苯并[a]芘的加合物和水解产物。对HPLC纯化产物的二极管阵列紫外、快原子轰击和在线大气压电离质谱分析表明，致突变性和致癌性更强的(+)-对映体在Asp(187)或Glu(188)处与蛋白质形成羧酸酯加合物，而(-)-对映体在His(146)处形成Nτ-组氨酸加合物。苯并[a]芘反式二醇环氧化物与人血清白蛋白反应的这种前所未有的对映体特异性，对于将加合物用作内暴露生物标志物的应用具有重要意义。
