E.s.r. of spin-trapped radicals in gamma-irradiated polycrystalline DL-alanine. A quantitative determination of radical yield

The quantitative aspects of determining free radicals in polycrystalline amino acids gamma-irradiated at room temperature and subsequently dissolved in spin-trap solutions were investigated. The deamination radical in DL-alanine was used for detailed studies and 2-methyl-2-nitrosopropane (MNP) was employed as the spin-trap. The spin-trapping efficiency (the number of radicals spin-trapped in solution divided by the number of radicals initially present in the gamma-irradiated solid) was found to be in the range 1 to 10 per cent for aqueous solutions depending on the experimental conditions. The effects of dose, particle size, pH, spin-trap concentration, age of spin-trap solution, MNP monomer to dimer ratio and the presence of organic solvents were investigated. Several reactions were found to decrease the spin-trapping efficiency; radical-radical recombination, the competition between the spin-adduct and the spin-trap for radicals and the reaction of radicals with the MNP dimer. The reaction of intact DL-alanine molecules with deamination radicals to produce H-abstraction radicals which are not spin-trapped does not significantly lower the spin-trapping efficiency. The results obtained with compounds such as glycine, glycylglycine, L-valine and L-proline suggest that the low spin-trapping efficiency found for DL-alanine may be representative of polycrystalline amino acids.     

Measurement of products from X-irradiated glycylglycine in oxygen-free aqueous solutions.

The product yields in X-irradiated aqueous solutions of glycylglycine (0.05 M and 1.0 M) were measured under deoxygenated conditions. Comparison was made between the results obtained from X- and 60Co gamma-irradiated glycylglycine solutions reported by Garrison, Sokol, and Bennett-Corniea (Radiat. Res. 53, 376-384, 1973). The mechanisms proposed by Garrison et al. were tested by evaluating the stoichiometric relationships. The two intermediate radicals, deamination and H-abstraction radicals, were produced in the initial interactions of glycylglycine with reactive species (e-aq, OH, H) formed in H2O. Although the difference was fairly large at 0.05 M, the production of deamination radicals agreed well with the consumption of the radicals at 1.0 M. The production and the consumption of H-abstraction radicals were within the estimated experimental error in dilute solutions. Among all the products only the G value of aspartic acid decreased with increasing concentration of glycylglycine. This could be attributed to the fact that more acetylglycine is formed at the expense of aspartic acid at 1.0 M than at 0.05 M glycylglycine solutions. Competitive reactions involved with deamination radicals under conditions of homogeneously distributed reactants are discussed to elucidate the radiation chemistry of glycylglycine.     

Stereochemistry and mechanism of reactions catalyzed by indolyl-3-alkane alpha-hydroxylase.

The reaction of tryptamine with indolyl-3-alkane alpha-hydroxylase is shown to remove stereospecifically the pro-S hydrogen at C-2 of the side chain and to give hydroxytryptamine of "R" configuration. The reaction therefore proceeds stereospecifically with net inversion of configuration at C-2 of the tryptamine side chain. In the reaction of L-tryptophan methyl ester, the enzyme also catalyzes stereospecific removal of the pro-S hydrogen at C-3, but the product 3-hydroxytryptophan methyl ester is racemic at C-3. The unreacted tryptophan methyl ester is shown to incorporate solvent hydrogen into the pro-S position at C-3 in an at least partially stereospecific manner, suggesting that the reaction of L-tryptophan methyl ester is reversible. The hydrogens at C-1 of the tryptamine side chain and the alpha-hydrogen of L-tryptophan methyl ester are shown to be retained in the reactions. The results support the notion that the enzyme catalyzes stereospecific 1,4-dehydrogenation of 3-substituted indoles to the coresponding alkylidene indolenines as the primary reaction, followed by stereospecific or nonstereospecific hydration of these intermediates as a secondary process. Substrate specificity studies with a number of tryptophan analogs are in excellent agreement with such a mechanism.     

Studies of short-lived radicals in the gamma-irradiated aqueous solution of uridine-5'-monophosphate by the spin-trapping method and the liquid chromatography.

An aerated aqueous solution of uridine-5'-monophosphate was gamma-irradiated with 2-methyl-2-nitrosopropane as a spin-trapping reagent. Liquid chromatography was applied to separate the stable nitroxide radicals in the irradiated solution. The radicals were detected by U.V. and e.s.r. spectrometry. The e.s.r. detection showed four peaks in the chromatogram. The orcinol method for detection of the residual sugar moieties was applied before and after reduction of the base to determine the existence of the 5,6-double bond for the molecules in each fraction. From the combined results of the e.s.r. and orcinol methods, the short-lived radicals which were trapped by 2-methyl-2-nitrosopropane were identified as radicals of N-1 and C-6 positions of the base moiety and t-butyl radical which was the radiolytic product of the trapping reagent.     

E.s.r. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids, N-acetyl amino acids and dipeptides

The radicals produced in several polycrystalline amino acids, N-acetyl amino acids and dipeptides by gamma-radiolysis at room temperature were investigated by spin-trapping. After irradiation in the solid state, the samples were dissolved in aqueous solutions f t-nitrosobutane and the trapped radicals identified by e.s.r. For alpha-amino acids, deamination radicals were found, and in some cases H-abstraction radicals were also observed. No decarboxylation radicals could be detected. For N-acetyl amino acids, except for N-acetylglycine, the major radical was the decarboxylation radical. For N-acetyglycine the H-abstraction radical from the glycine residue was observed. For dipeptides of the x-glycine, the radical formed by removal of H from the alpha-carbon of the carboxyl-terminal residue was always spin-trapped. Some primary deamination radicals and minor amounts of decarboxylation radicals could also be observed. For dipeptides of the type x-alanine, glycine-x and alanine-x, the decarboxylation radical was always the major spin-trapped radical. Some primary and secondary deamination radicals were also detected.     

Preparation of multiply deuterium-labeled cortisol.

Cortisol labeled with four deuterium atoms at chemically stable sites ([9,11,12,12-(2)H4]cortisol, cortisol-d4) was prepared by hydrogen-deuterium exchange and reductive deuteration reactions. After protecting the C-17 dihydroxyacetone side chain of cortisone (cortisone-BMD), hydrogen-deuterium exchange was carried out with 6.5% NaOD in MeOD, which was followed by protection of the C-3 carbonyl as the semicarbazone. Subsequent reductive deuteration at C-11 with NaBD4 followed by removal of exchangeable deuterium under the same exchange-reaction conditions in a medium of 6.5% NaOH in MeOH and deprotection afforded the desired cortisol-d4 with high isotopic content (d3, 21.2%; d4, 78.1%; d5, 0.74%). The method was applied to the synthesis of cortisol labeled with nine deuterium atoms [( 1,1,9,11,12,12,19,19,19-(2)H9]cortisol, cortisol-d9) starting from [1,1,19,19,19-(2)H5]cortisone (cortisone-d5).     

E.s.r. studies of halogenated pyrimidines in gamma-irradiated alkaline glasses.

The reactions of mobile electrons (em-) and oxygen radical anions (O--) with halogenated bases and nucleosides have been studies in gamma-irradiated alkaline glasses by e.s.r. and specific halogen-ion electrode techniques. It is shown that electrons react with halogenated uracil bases (XUr where X = Cl, Br. I but not F) by dissociative electron attachment to form uracil-5-yl radicals (U-) and halogen anions. The relative rates of reaction of em- with XUr decrease in the sequence BrUr greater than ClUr greater than FUr greater than IUr. Thermal annealing studies carried out on U- in H2O and D2O matrices support the hypothesis that U- in H2O hydrates across the 5-6 double bond in the temperature region 135 degrees-155 degrees K, and deuterates to a much smaller extent in D2O at temperatures above 155 degrees K. Studies on bromouridine and bromodeoxyurinde suggest that em- reacts with the base moieties to form U- type radicals which abstract H- from the sugar moieties of adjacent nucleosides.     

Effects of ATP and ADP on hydrogen-deuterium exchange in heavy meromyosin. An examination of difference spectra.

The exchange reaction of peptide hydrogens with deuterium has been followed by measuring the decrease of the amide II band for heavy meromyosin (HMM). The difference spectra between HMM and HMM + ATP, between HMM and HMM + ADP, and between HMM + ATP and HMM + ADP have been examined as functions of time in order to detect small differences in the kinetic behavior of these different states of HMM. It has been found that, at 14 degrees C and 26 degrees C (pH 8.0), the exchange reaction is slightly slower for HMM + ATP than for HMM, and slightly slower for HMM + ADP than for HMM + ATP. This indicates that the secondary structure of HMM changes its flexibility during the ATP splitting cycle.     

Carbon-13 nuclear magnetic resonance studies of adenosylcobalamin and alkylcorrinoids, selectively enriched with carbon-13.

The carbon-13 nuclear magnetic resonance spectra of a series of alkylcorrinoids, selectively enriched with 13C in the alkyl ligand, were recorded at 25.2 MHz and 25 degrees. The nature of the axial ligands markedly affects the chemical shift of the labeled alkyl moiety (trans effect) as well as the 13C resonances of selected carbon atoms of the corrin ring (cis effect). Although a number of factors appear to influence the trans effect on the chemical shift of the alkyl ligand (important among them being electric field effects), the cis effect appears to be dominated by changes in charge density (at the methine bridge carbon atoms, C-5, C-10, C-15) and by steric effects (at the methyl groups at C-1, C-5, and C-15) accompanying axial ligation. Spin-latice relaxation times of several organocorrinoids, selectively labeled with 13C in the ligands attached to cobalt, were also measured. The T1 values of the methylene carbons of [5'-13C]adenosylcobalamin and [2-13C]carboxymethylcobalamin are very similar to that of the methine bridge carbon atom C-10 of the corrin ring, indicating that rotation about the carbon-cobalt bond of these two corrinoids is severely restricted. On the other hand, internal rotation about the carbon-cobalt bond of methylcobalamin is rapid.     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. The source of the third methyl hydrogen in the 5'-deoxyadenosine generated from the cofactor during catalysis.

Ethanolamine ammonia-lyase is an adenosylcobalamin-dependent enzyme which catalyzes the conversion of ethanolamine and propanolamine to ammonia and the corresponding aldehydes. A mechanism has been proposed for this and other adenosylcobalamin-dependent reactions which involves cleavage of the carbon-cobalt bond of the cofactor followed by abstraction of a substrate hydrogen atom by the adenosyl fragment to form 5'-deoxyadenosine. In support of this proposal, a previous study demonstrated that the deamination of propanolamine by ethanolamine ammonia-lyase is accompanied by the reversible cleavage of the carbon-cobalt bond of the cofactor, with the production of 5'-deoxyadenosine (Babior, B.M., Carty, T.J., and Abeles, R.H. (1974) J. Biol. Chem. 249, 1689-1695). The present study is concerned with the origin of the third hydrogen atom on the methyl group of the 5'-deoxyadenosine produced in that reaction. The 5'-deoxyadenosine isolated from an incubation mixture initially containing enzyme, [5',5'-D2]adenosylcobalamin, and [1,1-D2]propanolamine was chemically degraded so that the 4' and 5' carbon atoms were, respectively, converted to the carbonyl and methyl carbons of acetaldehyde. Analysis of the p-nitrophenylhydrazone of the acetaldehyde by gas-liquid chromatography-mass spectroscopy revealed 3 deuterium atoms/molecule, indicating that two of the methyl hydrogens originated from adenosylcobalamin and the third was donated by substrate. This observation provides further support for the participation of 5'-deoxyadenosine in the mechanism of adenosylcobalamin-dependent reactions.     

Comparison between the unfolding rate and structural fluctuations in native lysozyme--effects of denaturants, ligand binding, and intrachain cross-linking on hydrogen exchange and unfolding kinetics

The hydrogen-exchange reactions of peptide NH groups in lysozyme were studied by the change in the intensity of the amide II band in the ir spectrum. The slowest exchanging hydrogens, which are involved in intramolecular hydrogen bonding, are further divided into two groups at lower temperatures; half of them are exchanged through local unfolding and the other half through major cooperative unfolding. In order to study the correlation of the change in hydrogen-exchange rates with the change in the unfolding rate constant, we observed the effects of intrachain cross-linking, the addition of denaturant and ligand binding on the exchange rates through local unfolding. Although the exchange rate through major unfolding is greatly decreased by intrachain cross-linking between Glu 35 and Trp 108 (1/22000), the exchange rate through local unfolding is only slightly decreased (1/20). Even at higher temperatures, where most intact lysozyme molecules unfold, the folded conformation of cross-linked lysozyme remains compact, and no intermediate exists in which many side-chain atoms are packed loosely so that the hydrogen-exchange reaction occurs rapidly. Neither the addition of 2-PrOD molecules nor (NAG)₃ binding affects the exchange rates through local unfolding. Our experiments confirm that the change in the unfolding rate constant does not correlate with the change in fluctuations in the relatively flexible hydrogen-bonded structure through which the exchange of peptide hydrogens takes place.     

Stereochemistry of the enolization of scytalone by scytalone dehydratase

In D(2)O, scytalone exchanges its two C2 hydrogen atoms for deuterium atoms at different rates. At pD 7.0 and 25 degrees C, half-lives for the exchanges are 0.8 and 10 days for the pro-S and pro-R hydrogens, respectively. The differential exchange rates allow for the preparation of multiple scytalone samples (through incubation of scytalone in D(2)O and then back exchanging with H(2)O) having differential levels of deuterium enrichment at the C2 pro-S and pro-R positions. From these samples, the stereochemical preference for hydrogen abstraction during the dehydration reaction mediated by the enzyme scytalone dehydratase was determined. At pH 7. 0, deuterium at the pro-S position has little effect on enzyme catalysis, whereas deuterium at the pro-R position produces kinetic isotope effects of 2.3 (25 degrees C), 5.1 (25 degrees C), and 6.7 (6.8 degrees C) on k(cat), k(cat)/K(m), and the single-turnover rate, respectively. The results are fully consistent with the enzyme catalyzing a syn elimination through an E1cb-like mechanism. The syn elimination is compatible with the interactions realized between a scytalone boat conformation and key active site residues as modeled from multiple X-ray crystal structures of the enzyme in complexes with inhibitors.     

Rates of spontaneous disintegration of DNA and the rate enhancements produced by DNA glycosylases and deaminases

To estimate the relative importance of alternate routes of spontaneous degradation of DNA and the rate enhancements produced by enzymes catalyzing these reactions, rate constants and thermodynamic activation parameters for the degradation of 2'-deoxynucleosides at 25 degrees C were determined by extrapolation of rates observed in the temperature range between 90 and 200 degrees C in neutral phosphate buffer. Rates of deamination of 2'-deoxycytidine, 1-methylcytosine, and cytidine were found to be identical within experimental error (t1/2 approximately 20 years, 37 degrees C). Rate constants for deamination of 2'-deoxyadenosine and 2'-deoxyguanosine, which could not be determined directly because of rapid glycoside cleavage, were estimated by assuming that methyl replacement should generate reasonable model substrates. The rates of deamination of 9-methyladenine and 9-methylguanine were found to be similar to each other (t1/2 approximately 6000 years, 37 degrees C) and approximately 10(2)-fold slower than the rates of glycoside cleavage in 2'-deoxyadenosine and 2'-deoxyguanosine. The deamination of 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine led to accelerated rates of glycoside cleavage. In the exceptional case of 2'-deoxycytidine, deamination and glycoside hydrolysis proceed at very similar rates at all temperatures. Glycoside cleavage proceeds with half-times ranging from 4 years for 2'-deoxyinosine to 40 years for 2'-deoxycytidine (37 degrees C). The rate enhancements produced by DNA glycosylases, estimated by comparison with the rates of these uncatalyzed reactions, are found to be substantially smaller than those produced by deaminases and staphylococcal nuclease.     

P.m.r. spectroscopy of dimethyl ethers of D-galactopyranose and its derivatives

P.m.r. parameters (determined at 100 MHz for solutions in deuterium oxide) are presented for di-O-methyl derivatives of D-galactopyranose (ten), methyl D-galactopyranoside (ten), and galactitol (five). The effects, on the methoxyl and anomeric-proton chemical-shifts, of anomeric change, methylation of neighboring hydroxyl groups, and change in configuration of adjacent carbon atoms bearing hydroxyl or methoxyl groups (other than at C-1) are discussed.     

Structure and thermal interconversion of cyclobilirubin IX alpha.

One of the two main photoproducts in bilirubin metabolism during phototherapy in neonatal hyperbilirubinaemia is (EZ)-cyclobilirubin. However, it has not yet been possible to come to a final conclusion as to its chemical structure, despite the fact that much effort has been expended on the problem. The present paper demonstrates that (EZ)-cyclobilirubin is formed by the intramolecular cyclization of the C-3-vinyl group with the position at C-7 rather than at C-6, without delta-lactone-ring formation. The evidence comes from 13C-n.m.r. spectra, which indicate that an oxygen-bound quaternary carbon atom is not present, and from 1H-n.m.r. spectra, which indicate that the orientation of the methyl group at C-2 is equatorial; these findings are supported by mass spectra. The existence of both an epimeric relationship at C-7 between (EE)- and (EZ)-cyclobilirubins A and B and of steric isomers of the hydrogen atom and methyl group at C-2 is supported by the fact that the methyl-group protons at C-2 and C-7 are observed as a paired signal in 1H-n.m.r. spectra, and that new signals at C-7, C-2 and C-3 beta appear in 13C-n.m.r. spectra, that mass spectra of (EZ)-cyclobilirubins A and B are extremely similar and that, furthermore, thermal interconversion between (EE)- and (EZ)-cyclobilirubins A and B is observed.     

Biosynthesis of mevinolin, a hypocholesterolemic fungal metabolite, in Aspergillus terreus

Mevinolin and compactin are fungal metabolites which inhibit cholesterol biosynthesis in mammalian systems. Biogenetically, mevinolin is formed from polyketide chains, one 18-carbon and one 4-carbon, derived from acetate in normal head to tail fashion. The remaining two carbons in mevinolin, namely C-2' and C-6 methyl groups, are transferred from S-adenosylmethionine. To distinguish the timing and sequence of these two methylation steps, [Me-14C]- and [Me-3H,14C]-L-methionine were fed to Aspergillus terreus at several selected production intervals. Location and distribution of labels were determined by the specific chemical degradation methods. The results have demonstrated clearly that transfer of methyl groups from two S-adenosylmethionine molecules to the biosynthetic precursors of mevinolin was a sequential process. Methylation at C-6 preceded that at C-2' of mevinolin. Both methylation steps proceeded with complete retention of hydrogens. Methyl groups were probably transferred to the anion-like intermediates.     

Site-specific OH attack to the sugar moiety of DNA: a comparison of experimental data and computational simulation.

Little computational or experimental information is available on site-specific hydroxyl attack probabilities to DNA. In this study, an atomistic stochastic model of OH radical reactions with DNA was developed to compute relative OH attack probabilities at individual deoxyribose hydrogen atoms. A model of the self-complementary decamer duplex d(CCAACGTTGG) was created including Na(+) counter ions and the water molecules of the first hydration layer. Additionally, a method for accounting for steric hindrance from nonreacting atoms was implemented. The model was then used to calculate OH attack probabilities at the various C-H sites of the sugar moiety. Results from this computational model show that OH radicals exhibit preferential attack at different deoxyribose hydrogens, as suggested by their corresponding percentage solvent-accessible surface areas. The percentage OH attack probabilities for the deoxyribose hydrogens [1H(5')+2H(5'), H(4'), H(3'), 1H(2')+2H(2'), H(1')] were calculated as approximately 54.6%, 20.6%, 15.0%, 8.5% and 1.3%, respectively, averaged across the sequence. These results are in good agreement with the latest experimental site-specific DNA strand break data of Balasubramanian et al. [Proc. Natl. Acad. Sci. USA 95, 9738-9742 (1998)]. The data from this stochastic model suggest that steric hindrance from nonreacting atoms significantly influences site-specific hydroxyl radical attack probabilities in DNA. A number of previous DNA damage models have been based on the assumption that C(4') is the preferred site, or perhaps the only site, for OH-mediated DNA damage. However, the results of the present study are in good agreement the experimental results of Balasubramanian et al. in which OH radicals exhibit preferential initial attack at sugar hydrogen atoms in the order 1H(5')+2H(5') > H(4') > H(3') > 1H(2')+2H(2') > H(1').     

Zinc release from irradiated yeast alcohol dehydrogenase

The release of Zn2+ from gamma-irradiated yeast alcohol dehydrogenase has been measured using atomic absorption spectrometry. Radiolysis is accompanied by release of Zn2+ at a rate which is dependent on the nature of the free radicals available for reaction. Hydroxyl radicals and hydrogen atoms readily cause zinc release with G values of 0.13 and 0.11 (/100 eV) respectively, whereas hydrated electrons are considered not to contribute to the demetallization process. The radiolytically generated radical anions I2-., (SCN)2-. and Br2-. enhance the rate of zinc release. Evidence is presented that the enzyme is demetallized as a result of free radical reactions at cysteine and histidine residues.     

Hydrogen exchange kinetics of nucleic acids: Double and triple helices with Hoogsteen-type basepairs

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid)·poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine·uracil basepair has been found to exchange at a measurably slow rate, 0.023 s^?1 (at 0°C), which is, however, much greater than that for a double-helix with the Watson-Crick type A·U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU)·poly(rA)·poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U·A·U base triplet have been found to exchange at a relatively slow rate, 0.0116 s^?1 (at 0°C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U·A·U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.     

Reactions of the hydrated electron with pyrimidine nucleosides halogenated at the sugar moiety: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by the reactions of hydrated electrons with pyrimidine nucleosides halogenated at the sugar moiety (2'-chloro-2'-deoxyuridine and 2'-chlorothymidine) were studied by e.s.r. and spin-trapping. 2-Methyl-2-nitrosopropane was used as the spin-trap. The usual spin-trapping technique was extended to frozen and deoxygenated systems to avoid contamination of the trapped radicals with side-products by spin-trapping 2-methyl-2-nitrosopropane itself. When this method was applied to 2'-chloro-2'-deoxyuridine, a free radical at the C-2' position of the sugar moiety was spin-trapped together with a free radical at the C-5 position of the base moiety. This indicates that hydrated electrons both add to the base moiety and eliminate halogen anions from the halogenated sugar moiety. In the case of 2'-chlorothymidine, however, only a free radical attributed to H-addition at the C-6 position of the thymine base was observed. No radicals produced by the reaction of hydrated electrons with the halogenated sugar could be spin-trapped.