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Chemical derivatization is useful for improving the ionization characteristics of poorly or nonionizable analytes in liquid chromatography--mass spectrometry (LC-MS). Dansyl chloride has been widely used as a derivatizing reagent for fluorescence detection and for facilitating the MS detection of phenols and amines, but not for general alcohols. A new dansylation method for improving the mass spectral sensitivity of unactivated alcohols was developed. The dansylated derivative was formed after incubation of the test compound cholesterol and excess dansyl chloride in C[H.sub.2][Cl.sub.2] in the presence of 4-(dimethylamino)-pytidine (DMAP) plus N,N-diisopropylethylamine at 65[degrees]C for 1 h, with an overall yield of 96%. The versatility of dansylation was investigated by utilizing representative lipid compounds (containing different numbers of hydroxy groups) for dansylation. All dansylated derivatives of the selected compounds were detected by LC-MS/MS in the electrospray ionization (ESI) positive ion mode. Validation of the method was established in terms of the sensitivity, stability, and repeatability of dansylation. The method was then applied to characterizing the P450 7A1 oxidation product (dansylated 7[alpha]-hydroxycholesterol) in human liver extracts using an LC-MS metabolomics/isotopic labeling approach (Tang, Z.; Guengerich, F. P. Anal. Chem. 2009, 81, 3071-3078). The dansylated derivative of the product was identified, with the signal increased by [10.sup.3]-fold compared with a previous method (derivatization with succinic anhydride and ESI negative ion MS). Quantitation of testosterone in human liver extracts was also done as an example of the application of the dansylation method. Thus, dansylation is a potential method of modifying many alcohols for detection by fluorescence and LC-MS analysis. 10.1021/ac1015497

One of the central problems in biochemistry in the postgenomic era is the elucidation of functions of proteins, including 'orphan' human cytochromes P450 (P450s), when the substrates are unknown. A general strategy for identification of endogenous substrates of P450s in tissue extracts using metabolomic and isotopic labeling approaches is described, involving four main steps: (1) In vitro incubation of a P450 enzyme system with cofactor and tissue extract is done under a mixture of [sup.18][O.sub.2]/[sup.16][O.sub.2] (1:1). (2) Liquid chromatography/mass spectrometry (LC/MS) assay of an organic extract of the reaction mixture is performed. (3) The isotopic labeling products appearing as M/M + 2 doublets can be directly identified using the program DoGEX (Sanchez-Ponce, R.; Guengerich, F. P. Anal Chem. 2007, 79, 3355-3362). (4) Characterization of potential candidates is done. Validation of the strategy was established using human P450 7A1 as an initial model to identify its known product, 7[alpha]-hydroxycholesterol, in liver extracts. The strategy was then applied to human P450s 1A2, 2C8, and 2C9 in untargeted substrate searches with human liver extracts. A total of seven fatty acids were identified and verified as substrates of these three hepatic P450s. The products were subsequently characterized as hydroxylation and epoxidation derivatives of fatty acids, using gas chromatography/mass spectrometry (GC/MS) analysis. Finally, kinetic studies were performed to confirm that the fatty acids are oxidized by P450s 1A2, 2C8, and 2C9. Thus, this strategy has been demonstrated to be useful in identifying reactions in tissue extracts with orphan human P450s.

A Matlab-based computer program termed Discovery of General Endo- and Xenobiotics (DoGEX) was developed, which uses wavelets and morphological analysis to process liquid chromatography--mass spectrometry (LC--MS) data. The output of the program is a list of integration areas as a function of retention time and molecular mass. A feature of the computer program is spectral filtering to facilitate the detection of chromatographic peaks with a particular isotopic ratio. The program DoGEX was used to automatically select oxidation products formed from felodipine (i.e., two chlorine atoms) and bromocriptine (one bromine atom) with cytochrome P450 3A4. The recognized isotope ratio can be changed to permit a natural or artificial mixture of isotopes to be monitored for selections. This computer program can be used to analyze LC-MS data for untargeted metabolic profiling experiments, e.g., to assign endogenous functions to newly characterized cytochrome P450 enzymes. In a representative example, an incubation of testosterone, NADPH, and a 1:1 [sup.16][O.sub.2]/[sup.18][O.sub.2] mixture yielded products with M and M + 2 ions resembling bromine doublets. Another use of the program is the subtraction of one set of [t.sub.R], m/z data from another, e.g., in comparisons of changes in patterns during enzyme reactions.

The recognition of specific DNA sequences by DNA-binding proteins is a crucial regulatory step in cellular processes such as DNA replication, control of gene expression, and the response to and repair of DNA damage. EcoRI restriction endonuclease is employed as a model for the interaction of DNA binding proteins with adducted DNA substrates.

Results demonstrate that there is no basic nitrogen atom involvement in the high-affinity binding of cytochrome P450 2D6 to substrates. Data indicate that some substrates bind through a negative charge either by electrostatic interaction or by hydrogen-bonding while others do not share this property.

The kinetics of incorporation of DNA-carcinogen adducts by polymerases are examined. Results show that polymerase stalling at 0(sup)6-alkylguanine changes the reaction kinetics, suggesting that an additional polymerase complex is involved.

Research reveals that both T7 DNA polymerase exo(sup)- and HIV-1 reverse transcriptase extend the 0(sup)6-methylguanine, while the latter extends 0(sup)6-benzylguanine additionally. Pre-steady-state kinetics indicate that neither dCTP nor dTTP insertion is favored during polymerization of 0(sup)6-benzylguanine containing DNA as opposed to 0(sup)6-methylguanine polymerization.

Steady-state kinetics of the Type I and Type II heme spectra and competition binding studies of the rabit liver cytochrome P450 1A2 show two binding sites for 1,4-phenyldiisocyanide and the alkyl 4-nitrophenyl ethers. The dissociation constants of these heterogeneous complexes further lend support for the two binding site model.

Group substitutions in the substrate analogue of human cytochrome P450 1A2, the p-alkoxyacylanilide, alter the rates of oxidations by approximately 5-fold and also improve the coupling efficiency from 6% to 38%. The rate contributions from individual steps in the catalytic cycle to the steady-state kinetics are discussed.

Results demonstrate that the rate-determining steps in the oxidation of phenacetin by the human cytochrome P450 1A2 enzyme, occur before the product formation as shown by substrate recognition sequence mutagenesis. The rate-determining steps do not occur until a 800-fold turnover of the catalytic activity of the enzyme.

Research has been conducted on the cytochrome P450 3A4. Result indicate marked catalytic regioselectivity of the enzyme and unusual heterotropic and homotropic cooperativity patterns.

Randomized human cytochrome P450 plasmid libraries were screened for mutant 1A2 enzymes using the Escherichia coli mutagenicity tester strain DJ3109 pNM12. The strain expresses Salmonella N-acetyltransferase and contains a lacZ reporter allele, and shows that the profiles of the P450 mutants differed significantly for three reactions. All Phe-226 mutants had substantially reduced activity, in contrast with Glu-225 mutants which displayed increased catalytic activities.

Most of the pre-steady-state kinetic studies of HIV-1 reverse transcriptase (HIV-1 RT) have shown substoichiometric bursts of product formation in the initial reaction cycle. The extinction coefficient of the HIV-1 RT heterodimer was altered by applying quantitative amino acid analysis. It was observed that normal nucleotide incorporation occurs with quantitative bursts in the first cycle. Findings showed that HIV-1 RT is fully active in normal nucleotide incorporation and that substoichiometric bursts with altered systems are well-defined by the presence of nonproductive ternary complexes, which can isomerize to productive complexes.

The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) incorporation into DNA by Escherichia coli polymerases I exo- (KF-) and II exo- (Pol II-), HIV-1 reverse transcriptase (HIV-1 RT) and bacteriophage T7 exo- (T7-) were investigated. The goal was to examine the misincorporation potential for 8-oxo-dGTP and to study the function of base pairing symmetry in DNA polymerase fidelity. Results indicated that polymerase fidelity does not rely only on the overall geometry of Watson-Crick base pairs and the enzyme binding site is asymmetrical.

A study was conducted to examine the influence of phospholipids in the membrane binding and insertion of the microsomal protein rabbit cytochrome P450 1A2 into phospholipid bilayers. P450 1A2 was determined from the liver microsomes of rabbits treated with 5,6-benzoflavone. Results indicated the importance of acidic phospholipid negative charges on the initial binding of P450 to membranes.

A study was conducted to examine the modification of nucleotides opposite five-membered exocyclic ring guanine derivatives by Escherichia coli polymerases. A modified Cary 14/OLIS system was used to record ultraviolet spectra while reagents were purified from recombinant expression vectors. Results showed differences in the way the five-membered exocyclic adducts were processed by polymerases.

A study was conducted to examine the 8-oxo-7,8-dihydroguanine base pair mismatches of a Bacteriophage when replicated in the bacterium's DNA polymerase. The mismatched occurred when 8-oxo-7,8-dihydroguanine had base pair with cytosine or adenine. Results suggested that the mispaired system could be attributed to discrimination in extension which resulted from the conformational change rates rather than in rates of phosphodiester bond formation.

The exo isomer of aflatoxin [B.sub.1] (AF[B.sub.1]) 8,9-epoxide appears to be the only product of AF[B.sub.1] involved in reaction with DNA and reacts with the [N.sup.7] atom of guanine via an [S.sub.N]2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in [H.sub.2]O (0.6 [s.sup.-1] at 25 [degrees] C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA ([K.sub.d] = 0.43 mg [ml.sup.-1], or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 [s.sup.-1]. Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 [s.sup.-1] and half-maximal velocity at a DNA concentration of 1.8 mg [ml.sup.-1] (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 [s.sup.-1] at pH 6.2 with 0.17 mg DNA [ml.sup.-1]). The same rate enhancement effect was observed with poly[dA-dT][center dot]poly[dA-dT], in which the epoxide can intercalate but not form significant levels of [N.sup.7] purine adducts, and with single-stranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.

Steady-state and pre-steady-state kinetics were used on the replicative T7 polymerase exo- and human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) to determine the kinetic differences among DNA polymerases that share similar structures and mechanisms of actions. Results indicate that the kinetic differences among the DNA polymerases involve conformational changes prior to chemistry (k3) and chemistry (k4). Furthermore, results reveal that both k3 and k4 contribute to the rate-limiting phase during incorporation of dCPT and dATP opposite 8-oxoGua.

Oligomers containing 1,N2-epsilon-Gua and HO-ethanoGua were synthesized based on a novel strategy involving the incorporation of the DNA adducts into defined oligonucleotides. The closed form of HO-ethanoGua contained different H2O elements compared to the 1,N2-epsilon-Gua DNA adduct. Oligomers containing HO-ethanoGua also exhibited a tendency to produce a ladder of products that were extended by one base at a time. Furthermore, both 1,N2-epsilon-Gua and HO-ethanoGua exhibited miscoding potentials.

The method for the estimation of conjugation rates with aflatoxin B1 (AFB1) exo-8,9-epoxide with subtrates that are unstable to H2O was developed based on the determination of stable hydrolysis and conjugation products. The method involved the rigorous quantitation of the rate of nonenzymatic degradation of AFB1 exo-8,9-epoxide in H2O and subsequent matching of results with a mathematical model of the kinetic scheme. The method permitted the measurement of reaction completion under conditions that do not permit the direct observation of conjugation.

P450 3A4 reconstitution systems were developed to examine a number of substrates that have been previously observed to demonstrate positive cooperativity. Several P450 3A4 substrates showed cooperative interactions, including testosterone, 17(beta)-estradiol, amitriptyline and most notably aflatoxin. Some catalytic activities were stimulated by the flavonoid 7,8-benzoflavone.

The biochemical activity of sulfaphenazole was analyzed during the catalytic activities of recombinant human liver cytochromes P450 in yeast. The inhibition of CYP 2C9 recombinant cytochrome P450 activity by sulfaphenazole involves the bonding of three nitrogen atoms from sulfaphenazole with CYP2C9-Fe(III). The NH2 group of sulfaphenazole exhibited high affinity towards CYP 2C9 iron while the N-phenyl group of the compound also formed a hydrophobic bond with the amino acid residues in the active site of CYP 2C9.

The kinetics and dynamics underlying the hydrolysis of the mycotoxin aflatoxin B1 (AFB1) were studied. Specifically, the hydrolysis rate of AFB1 exo-8,9-epoxide was determined with respect to the pH of H2O. The results, which indicated the genotoxic isomer's fast and spontaneous reaction, revealed the reversible conversion of the hydrolysis product, AFB diol, to the oxyanionic AFB1 alpha-hydroxydialdehyde under moderately basic conditions.

Analysis of the N-terminal sequence MALLLAVFL... of mammalian cytochrome P450, and random mutagenesis in this region shows that the leucine triplet at 3-5 position is sufficient to block the action of deformylase and N-terminal Met aminopeptidase, thus leaving the N-terminal blocked. Mutant P450, incorporated into spheroplasts, is not digested by proteinase K. This indicates that the N-terminus is imbedded in the membrane, thus blocking the N-terminal processing. Direct inhibition of the action of the processing enzyme can be the other possibility.

Steady state analysis reveals that the rate of incorporation of dCTP is faster than that of dATP on the 8-oxo-7,8-dihydroguanine (8-oxoGua) site of a DNA template using Escherichia coli DNA polymerases I exo(super -) and II exo(super -) as enzymes. Pre steady state kinetic plots of the rate of incorporation of dCTP-alpha-S and dATP-alpha-S phosphorothioate analogs suggests that the formation of a phosphodiester bond controls the rate of incorporation of A and C opposite 8-oxoGua in a polymerization reaction. These insertion reactions bring about mutations in the DNA molecule.

The mechanisms of polycyclic hydrocarbon oxidations of 9-methylanthracene to quinones in several systems were investigated. Reactions in rat liver microsomes resulted in the formation of dihydrodiols from 9-methyl- and 9-ethylanthracenes while products formed in cytochrome P450 2B1 were dependent on oxygen levels. The horseradish peroxidase system resulted in the formation of three oxidation products, whose amounts decreased when oxygen was omitted from the system. The results suggest that the 9-methylanthracene radical cation may have been involved in the P450 and FeTPP pathways, but evidence supporting one-electron oxidations of the polycyclic hydrocarbons is lacking.

A study was conducted to determine the role of cytochrome P-450 in the synthesis of epoxy alcohols from fatty acid hydroperoxides in rat liver microsomes. The result revealed stereoselective formation of epoxy alcohols from 15R- and 15S-hydroperoxyeicosatetraenoic acids, indicating the involvement of P-450. This means that enzymatic catalysis, aside from non-enzymatic catalysis, is involved in the formation of epoxy alcohols from fatty acid hydroperoxides in mammalian tissues.

The region immediately beyond the conserved Cys region, a putative axial ligand to the heme, is a common epitope for P450 2C10 and P450 2E1 and is also a common epitope for the immunochemical cross-reactivity observed with some other P450 proteins. This was determined after the identification of a common cytochrome P450 epitope through an analysis by protease digestion and Edman degradation. From the intensity of the immunoblotted peptides, the epitope in the C-terminal region near the conserved heme-binding region seems to be a major one.

Electron transfer from NADPH-P450 reductase to many acceptors is stimulated by divalent cations. Substrates and b5 are able to bind to P450 3A4 to affect its rate of reduction by the reductase. P450 3A4 catalyzes testosterone 6beta-hydroxylation within Escherichia coli cells. Catalyzation is also aided by spinach ferredoxin and NADPH-ferredoxin reductase. This supports the idea that events associated with P450 reduction and oxygen activation are commonly rate-limiting for oxidations catalyzed by P450.

Analysis of the general relative stabilities of oligomeric duplexes with different base pairs reveal that S-(2-N7-guanylethyl)GSH does not contribute to a higher frequency of T-misinsertion than G. Mutagenesis by N7-guanyl adducts involves ionization at the guanine-N1 position. Ionized base pairs take part in mispairing and mutagenesis.

The aflatoxin B1 (AFBI) exo epoxide isomer reacts with DNA through an S2 mechanism, resulting in specific trans ring opening of the isomer, whereas the endo isomer is inactive. Dymethyldioxirane-catalyzed oxidation or cytochrome p450 oxidase-catalysed enzymatic reactions yield the two AFB1 isomers. The results confirm that the exo epoxide is the potent mutagen in AFB1.

The expression of mammalian glutathione S-transferase 5-5 (GSHS) in Salmonella typhimurium resulted in bacterial susceptibility to genetic mutation, upon being treated with dihalomethanes. GSHS-transferase-mediated conjugation were responsible for the genotypic responses on the addition of dihalomethanes. Pretreatment of the transfected palsmid vector, consisting of the rat GSHS-transferase, with ethylene dihalides resulted in the production of base-pair revertants were observed. Liver tumor formation in mice might be associated with the GSH conjugation mechanism.

Experiments were conducted to modify cytochrome P450 1A2 enzymes via the mechanism-based inactivator 2-ethynylnaphthalene (2EN) and the photoaffinity label 4-azidobiphenyl. The results showed that 2EN efficiently inactivated rabbit P450 1A2 but did not inactivate human P450 1A2. In both P4501A2 enzymes only a lone tryptic peptide was labeled. The result also revealed the varying labeling patterns of the three sample P450s despite their having almost identical primary sequences.

The synthesis of etheno derivatives of nucleic acid bases from 1-substituted oxiranes is investigated via the reaction of N6-methyladenosin with 1-chlorooxirane to give the 7,8-dihydro-8-hydroxy-9-methyl-3-beta-D-ribofuranosylimidazo(2,1-i)purinium species. Results suggest a mechanism in which the most basic endocyclic nitrogen atom of adenosine or cytidine attacks the methylene of a substituted oxirane to give an N-(2-oxoethyl) adduct. Schiff base formation then closes the adduct to give the etheno adduct.

A comprehensive genetic, metabolomic and biochemical study on the catalytic properties of Streptomyces coelicolor cytochrome P450 (P450) 154A1, which has a unique heme orientation in its crystal structure, is described. The biotransformation of dipentaenone by P450 154A1 is an unexpected intramolecular cyclization to a Paterno-Buchi-like product, without oxidation/reduction.

The bypass of 3-(2'-deoxy-[beta]-D-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one ([M.sub.1]dG) by the human Y-family DNA polymerases k, l, and Rev1 is evaluated. The results have shown that DNA hPol k or the combined action of hPol l or Rev1 and hPol k has bypassed [M.sub.1]dG residues in DNA and has generated products that are consistent with some of the mutations induced by [M.sub.1]dG in mammalian cells.

The article presents a complete characterization of the structure of 1,[N.sup.2]-ethenodeoxyguanosine lesion (1,[N.sup.2]-[sigma]dG), which is shown to have no opposite dC. The biological processing of the discussed lesion is shown to be highly affected by the stabilization of the Dpo4 polymerase.

The interactions between individual amino acids and metal ions are delineated by individually mutating each of the acidic amino acid residues in human topoisomerase II[alpha] to either cysteine or alanine. The results have shown that the four acidic amino acid residues have interacted with metal ions in the DNA cleavage/ligation active site of topoisomerase II[alpha].

Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus is used as a model to conduct kinetic and structural studies and probe the mechanism by which translesion polymerases bypass 3-(2'-deoxy-[beta]-D-erythro-pentofuranosyl)pyrimido[1,2-[alpha]purin-10(3H)-one ([M.sub.1]dG). The result obtained is consistent with the mutagenicity of [M.sub.1]dG and demonstrated the possible effect of lesion on replication events.

The in vitro oxidation of many endogenously occurring etheno adducts and their corresponding 2'-deoxynucleosides is examined. The results have shown that multiple exocyclic adducts have undergone oxidation and glycolytic cleavage in vitro in rat liver cytosol and these multiple pathways of biotransformation has produced an array of products.

The ability of [O.sup.6]-alkylguanine-DNA alkyltransferase to repair interstrand cross-link DNA damage is investigated. The butane cross-links present in the oligodeoxiribonucleotides of DNA are found to hinder the repair mechanism.

A study of a chemical reaction between chloroethylene oxide, the ultimate carcinogen formed from vinyl chloride, and guanine (Gua) giving rise to 7-(2-oxyethyl)-guanine, the major formed is presented. This reaction represents an initial step of carcinogenesis associated with vinyl chloride.

It is shown that [Ca.sup.2+] is a cofactor for Y-family DNA polymerase IV (Dpo4)-catalyzed polymerization with both native and 8-oxoG-containing DNA templates. Both dNTP and ddNTP are substrates of the polymerase in the presence of either [Mg.sup.2+] or [Ca.sup.2+], but no pyrophosphorolysis occurs in the presence of [Ca.sup.2+].

The effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. The finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.

Several issues regarding the rate-limiting nature of individual reaction steps in catalysis by rabbit liver cytochrome P450 (P450) 1A2 were addressed using anisoles and other substrates. This indicates that increasing ground-state binding can increase catalytic efficiency but not k(sub cat), and suggest that nonproductive binding modes and abortive reduction of O2 are factors to attenuate catalytic efficiency.

Research indicates that the low pK(sub)a of 4-5 and high reactivity of Cys145 of the human omicron(sup)6-alkylguanine-DNA alkyltransferase are integral parts of the catalytic function, cross-linking reactions, DNA repair inhibition function of the enzyme. The low pK(sub)a is zinc-dependent.

Results show that amine substrates electrostatically interact with Glu216 in binding the repertoire of human cytochrome P450 2D6 ligands as revealed by mutagenesis analysis. Data indicate that the acidic amino acid residue Glu216 is associated with the substrate recognition sequence 2 and the F-helix.