Spectral Differentiation and Immunoaffinity Capillary Electrophoresis Separation of Enantiomeric Benzo(a)pyrene Diol Epoxide-Derived DNA Adducts

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• 作者：Beata Miksa ; Raja Chinnappan ; Nhan C. Dang ; Mike Reppert ; Brock Matter ; Natalia Tretyakova ; Nenad M. Grubor ; Ryszard Jankowiak
• 刊名：Chemical Research in Toxicology
• 出版年：2007
• 出版时间：August 2007
• 年：2007
• 卷：20
• 期：8
• 页码：1192 - 1199
• 全文大小：145K
• 年卷期：v.20,no.8(August 2007)
• ISSN：1520-5010

文摘

Antibody cross-reactivity makes separation and differentiation of enantiomeric analytes one of the most challenging problems in immunoanalytical research, particularly for the analysis of structurally related biological molecules [such as benzo(a)pyrene (BP) metabolites and BP-derived DNA adducts]. It has recently been shown that the interaction of enantiomers of BP tetrols (BPT) with a promiscuous anti-polycyclic aromatic hydrocabon (anti-PAH) monoclonal antibody (mAb) allowed for separation of all four enantiomeric isomers using immunoaffinity capillary electrophoresis [ Grubor N. M., Armstrong D. W., and Jankowiak R. (2006) Electrophoresis27, 1078] and unambiguous spectral resolution using fluorescence line narrowing spectroscopy (FLNS) [ Grubor N. M., Liu Y., Han X., Armstrong D.W., and Jankowiak R. (2006) J. Am.Chem. Soc.128, 6409]. Here, we expand the use of the above two methodologies to the group of biologically important molecules that are products of BP diol epoxide (BPDE)-induced DNA damage. Four diastereomeric anti-BPDE-derived deoxyguanosine (dG) adducts, that is, (+)- and (−)-anti-trans-BPDE–N²-dG and (+)- and (−)-anti-cis-BPDE–N²-dG, were electrophoretically separated and spectroscopically differentiated using 8E11 mAb raised against BP–DNA conjugates. In fluorescence line narrowing spectroscopy (FLNS) experiments, complexes of BPDE–dG adducts with mAb revealed differences in fluorecence origin band positions, bandwidths, and vibrational patterns for all four BPDE–N²-dG adducts. Narrow fluorescence origin bands observed for (−)-trans-BPDE–dG (70 cm⁻¹) and (+)-trans-BPDE–N²-dG (80 cm⁻¹) suggest spatial constraint within the mAb binding pocket. Broader origin bands observed for cis type adducts (~120 cm⁻¹) in 8E11 mAb suggest different binding geometries and/or conformational changes, as also indicated by changes in vibrational frequencies observed for the (+)-anti-cis and (−)-anti-cis adducts complexed with mAb. FLNS revealed that binding conformations and interactions within the mAb binding pocket are different for each adduct, enabling unambiguous positive identification. The methodologies described in this manuscript could also be used for analysis of DNA adducts following enzymatic hydrolysis of BPDE-adducted DNA to free nucleosides.