Chem. Res. Toxicol. 1992,5, 71-76 serine protease mechanism. J. Biol. Chem. 258, 59-66. (22) Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd ed., pp 405-413, W. H. Freeman, New York. (23) Bowman, B. T.,and Sans, W. W. (1983) Determination of Octanol-water partitioning coefficients (KJ of 61 organophosphorus and carbamate insecticides and their relationship to
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respective water solubility (S)values. J. Enuiron. Sci. Health B18,667-683. (24) Mundy, R. L.,Bowman, M. C., Farmer, J. H., and Haley, T. J. (1978) Quantitative Structure activity study of a series of substituted 0,O-dimethyl 0-(p-nitropheny1)phosphorothioatesand 0-analogs. Arch. Toxicol. 41, 111-123.
Human Serum Albumin-Benzo[ a Ipyrene anti-Diol Epoxide Adduct Structure Elucidation by Fluorescence Line Narrowing Spectroscopy Billy W. Day,+Jp§Mark M. Doxtader," Robert H. Rich,$ Paul L. Skipper,' Kuldip Singh,II Ramachandra R. Dasari,ll and Steven R. Tannenbaum*yt*$ Department of Chemistry, Division of Toxicology, and George R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received September 16, 1991 Cryogenic (4-10 K) laser-induced vibrationless ground state and vibronic excited state fluorescence emission spectra of the adducts resulting from reaction in vitro of human serum albumin and the carcinogen (f)-r-7,t-8-dihydroxy-c-9,c-lO-epoxy-7,8,9,lO-tetr~ydrobenzo[a]pyrene were recorded in order to determine the structures formed. Comparison of these fluorescence line-narrowed (FLN) spectra to those obtained from BaP-7,8,9,10-tetrahydrotetrols, synthetic N-t-BOC-alaninate ester, and N - and N"-histidine amine anti-BuPDE adducts revealed that a mixture of adduct types are formed with the protein. Extensive dialysis of the adducted protein simplified the FLN spectrum, causing it to become nearly identical to the FLN spectrum obtained from the stable peptide adduct. Comparison of the FLN spectra of the synthetic histidine adducts to those obtained from peptide adducts isolated from enzymic digestion of the adducted protein indicated that only one of the imidazole nitrogens is the nucleophile which forms a stable adduct with anti-BaPDE. The FLN studies confirm that "-histidine adducts are formed between human serum albumin and the C-10 position of anti-BaPDE.
Introduction Fluorescence line narrowing (FLN)' spectroscopy originally arose from the desire to obtain narrow-band vibrational excitation and emission spectra from molecular species which do not easily dinsolve in the paraffii solvents so successfully implemented as Shpol'skii matrices (1,2). An entirely different class of compounds from those studied by Shpol'skii spectroscopy is amenable to sensitive and selective analyses using FLN. Compounds in biological studies are usually only sparingly soluble in the hydrophobic alkanes and require polar solvents which adopt a much less defined orientational structure in the solid state than do the alkanes. At cryogenic temperatures the chromophores & p e d in the heterogeneous solvents used for FLN reside in a number of energetically inequivalent microenvironments or "sites". Because of the relative orientation of the solute within the solvent, each slightly different microenvironment imparts a specific energy to *Author 6 whom correspondence should be addressed at Room 56-309, Department of Chemistry, Division of Toxicology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. 'Department of Chemistry. Division of Toxicology. 8 Present address: Departments of Pharmaceutical Sciences and Environmental & Occupational Health, University of Pittsburgh, Pittsburgh, PA 15261. 11 George R. Harrison Spectroscopy Laboratory.
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the chromophore. A change of substituents on the same chromophore imparts additional differences in microenvironments. The result is a collection of chromophores arising from structurally similar molecules which may undergo the same nominal transition, but only at specific excitation energies corresponding to their various relationships to the host matrix. Narrow vibrational spectra are revealed in heterogeneous solvents only if narrow-band excitation, such as that originating from a laser, is used as a photon source. In addition, the temperatures ordinarily required to achieve narrow lines in paraffi matrices are not adequately low to reduce phonon contributions from the heterogeneous glass matrices used in FLN spectroscopy. While the ultimate solvent system for FLN has not been rigorously defined with respect to molecular dimensions, it is often possible to find suitable solvents which minimize electron-phonon coupling. FLN spectroscopy has been successfully applied to a variety of problems, including the analysis of coal tars (3) and petroleum distillates ( 4 5 ) and, because of its applicability to aqueous systems, the analysis of polar compounds in waste water (6). FLN has also been used to study fast kinetic processes (7,8) and the vibronic effects Abbreviations: BaP, benzo[a]pyrene; anti-BaPDE,r-7,t-8-dihydroxy-c-9,c-l0-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene; BaP tetrahydrotetrols, r-7,t-8,t-9,t-10and r-7,t-8,t-9,c-lO-tetrahydroxy-7,8,9,10tetrahydrobenzo[a]pyrenes; t-BOC, tert-butyloxycarbonyl; FLN, fluorescence line narrowing.
0893-228x/92/2705-0071$03.00/00 1992 American Chemical Society
Day et al.
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w.=n of deuteration in complex molecules (9). More recently, it has been shown that FLN can provide information concerning tryptophan residues in proteins (10). From our own perspective, the most significant advances which have Q L2 occurred concern the application of FLN to the study of chemical carcinogenesis and macromolecule-carcinogen Q L3 I adducts (2, 11, 12). With a minimum amount of effort applied to sample preparation, FLN has given detection Laser H i h Voltage MGiWChromBU sensitivities comparable to more traditional trace analytical Power supply Generator Generator methods such as gas chromatography-mass spectrometry, enzyme-linked immunosorbent assay, and 32P-postlabeling procedures. Its selectivity has provided powerful qualitative insight into the structures of xenobiotic-macroPlaner COmputK Mulbchannel molecule adducts including elucidation of the type of nucleophilic heteroatom involved in the adduction in vivo of human hemoglobin with the metabolically generated Figure 1. Schematic of the fluorescence line-narrowing speccarcinogen (*)-r-7,t-8-dihydroxy-c-9,c-lO-epoxy-7,8,9,10-troscopy experimental arrangement used in this study: s,sample; PDA, gated intensified photodiode array; L1,lO-cm focal length tetrahydrobenzo [a]pyrene (anti-BaPDE) (I2). We are quartz cylindrical lens; L2,5cm, and L3,15cm focal length quartz particularly interested in protein adducts of anti-BaPDE, biconvex lens. since its parent compound BaP is the environmental pollutant most often used as a benchmark for evaluation samples were dissolved in 5:4:1 glycerol-H,O-EtOH and transof contamination or exposure to polycyclic aromatic hyferred to 3 mm 0.d.-2 mm i.d. quartz tubes. Prior to insertion drocarbons. into the cryostat the samples were thoroughly degassed by soWe have previously been successful in correlating the nication at
