An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.     

E.s.r. and spin-trapping studies of the reactions of hydrated electrons with dipeptides.

The reactions of hydrated electrons (eaq-) with 55 dipeptides and 25 acetyl and formyl amino acids have been studied by e.s.r. and spin-trapping techniques. Gamma-radiolysis of deaerated aqueous solutions was used to generate eaq-, and sodium formate or t-BuOH was added to scavenge the OH radicals. t-Nitrosobutane was employed as the spin-trapping reagent. The radical,--CO---NH--, which is the initial product of the reactions of eaq- with dipeptides, was observed only for val-gly, val-ala, val-leu and ile-ala. For most of the dipeptides this radical converts to the primary deamination radical, CHR'-CONH-CHR-COO-, where R and R' are the side-chains of the common amino acids. In many cases a radical of the type CHR-COO-, formed by secondary deamination, was also observed. Only secondary deamination reactions were observed for dipeptides containing beta-alanine as the amino terminal residue and for acetyl and formyl amino acids. The secondary deamination reactions of eaq- with dipeptides, acetyl and formyl amino acids in aqueous solutions have not been observed previously. This type of reaction is of interest since it brings about main-chain scission in polypeptides and proteins.     

E.s.r. of spin-trapped radicals in aqueous solutions of peptides. Reactions of the hydroxyl radical.

The reactions of hydroxyl radicals with 30 dipeptides and several larger peptides were studied in aqueous solutions. The OH radicals were generated by U.V. photolysis of H2O2. The short-lived peptide radicals were spin-trapped using t-nitrosobutane and identified by e.s.r. For dipeptides containing the amino terminal residues glycine, alanine and phenylalanine, abstraction of the hydrogen from the carbon adjacent to the peptide nitrogen was the major process leading to the spin-adducts. Such radicals will be referred to as backbone radicals. Dipeptides with a carbonyl terminal serine residue and also glycylglutamic acid form both backbone and side-chain radicals, with the latter being formed in larger quantities. For dipeptides, side-chain radicals were detected on either the carboxyl or amino terminal residues of both. The effect of pD on the e.s.r. sectrum of the spin-adducts of glycylglycine was studied and the pK of the carboxyl group of this radical was determined to be 2.5. For (Ala)3 and (Ala)n, with an average value of n = 1800, backbone and minor side-chain radicals were observed. For ribonucleases-S-peptide, containing 20 amino acid residues, both backbone and side-chain radicals were detected.     

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.     

E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydroxyl radical.

The radicals produced by reactions of hydroxyl radicals with amino acids in aqueous solutions have been investigated. Hydroxyl radicals were formed by U.V.-photolysis of hydrogen peroxide and the short-lived amino acid radicals were spin-trapped by tert-nitrosobutane and identified by electron spin resonance spectroscopy. Nineteen amino acids were studied, and several radicals were identified which have not been observed previously by other methods. Only side-chain radicals were identified for alanine, threonine, aspartic acid, asparagine, lysine, phenylalanine, tyrosine, proline and hydroxyproline; whereas for glycine the C(2) carbon radical was spin-trapped. Both C(2) carbon radicals and side-chain radicals were assigned to valine, leucine, isoleucine, serine, glutamic acid, glutamine, arginine and methionine.     

E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydrated electron.

The reactions of hydrated electrons (eaq-) with amino acids were investigated by the spin-trapping method and by electron spin resonance. Tertiary nitrosobutane was used as a spin-trap to stabilize the short-lived radicals. Hydrated electrons were produced by gamma-radiolysis of de-aerated aqueous solutions of amino acids in the presence of sodium formate or tertiary butanol to scavenge OH. Radicals produced by reductive deamination of 19 amino acids were identified. Radicals formed by scission of the CH3-S- and -S-CH2- bonds of methionine as well as by deamination were observed. In the case of phenylalanine the radical formed by electron addition followed by proton transfer was identified. The reaction of proline and of hydroxyproline with eaq- resulted in the opening of the cyclic structure.     

E.s.r. study of spin-trapped radicals formed during the photolysis of aqueous solutions of acid amides and H2O2.

Free radicals formed by the reactions of OH radicals with amides and their N-methylated derivatives in aqueous solutions have been studied. The OH radicals were produced by U.V.-photolysis of H2O2, and the short-lived amide radicals were converted to more stable nitroxide radicals by addition to a spin-trap, tert-nitrosobutane. The spin-trapped radicals were identified by e.s.r. spectroscopy. For acetamide, chloroacetamide, malonamide, succinamide and propionamide, the observed radicals were formed by H-abstraction from the carbon atoms attached to the carbonyl group. The H atom attached to the carbonyl group was abstracted in formamide. For N-methyl acetamide, N,N-dimethyl acetamide and the corresponding formamide derivatives, H-abstraction occurred only from the N-methyl group. The non-equivalency of the amide protons was observed in the spin-trapped radicals for acetamide, formamide, malonamide, succinamide and propionamide. The identification of the site of OH attack on N-methyl amides is helpful for the study of radical formation in peptides and proteins.     

Photolysis of N-methyl and N-acetyl substituted amino acids

Summary Experiments on the production of free radicals in aqueous solutions of N-methyl and N-acetyl substituted amino acids by UV light are reported. The ESR spectra observed at 77 K show that the bond of the CH_3–group of these substances is ruptured to a great deal compared with other bond ruptures producing paramagnetic centres. But in N-methyl substituted amino acids the methyl radical is less predominant than in N-acetyl substituted ones. At temperatures higher than 200 K the ESR spectra of all substances studied in our experiments are similar. It is supposed that a radical is formed with an unpaired spin near an oxygen atom.A part of this paper was reported at a Congress in Freiburg i. Br. (Germany) organized by the Deutsche Gesellschaft für Biophysik, October 2–4, 1974.     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals

The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found.     

Catabolism of amino acids in bacteria of the genus Klebsiella

Propagation and activity level of 18 enzymes catalyzing deamination reactions of dicarboxylic and oxyamino acids and enzymes of amino acid reamination and amino acid N-acyl-derivatives' deacylation have been studied in Klebsiella bacteria. Klebsiella the most actively utilizes serin, threonine, aspartic and glutamic acids and aromatic amino acids. The first three amino acids are utilized by deamination, aromatic acids- in aminotransferase reaction with alpha-ketoglutaric acid, glutamic acid--by deamination and decarboxylation. Besides, Klebsiella actively deacylates N-acyl-derivatives of amino acids.     

Free Radical Formation by Ultrasound in Aqueous Solutions. A Spin Trapping Study

Our recent spin trapping studies of free radical generation by ultrasound in aqueous solutions are reviewed. The very high temperatures and pressures induced by acoustic cavitation in collapsing gas bubbles in aqueous solutions exposed to ultrasound lead to the thermal dissociation of water vapor into H atoms and OH radicals. Their formation has been confirmed by spin trapping. Sonochemical reactions occur in the gas phase (pyrolysis reactions), in the gas-liquid interfacial region, and in the bulk of the solution (radiation-chemistry reactions). The high temperature gradients in the interfacial regions lead to pyrolysis products from non-volatile solutes present at sufficiently high concentrations. The sonochemically generated radicals from carboxylic acids, amino acids, dipeptides. sugars, pyrimidine bases. nucleosides and nucleo-tides were identified by spin trapping with the non-volatile spin trap 3.5-dibromo-2.6-dideuterio-4-nitrosobenzenesulfonate. At low concentrations of the non-volatile solutes. the spin-trapped radicals produced by sonolysis are due to H atom and OH radical reactions. At higher concentrations of these non-volatile solutes, sonolysis leads to the formation of additional radicals due to pyrolysis processes (typically methyl radicals). A preferred localization of non-volatile surfactants (compared to analogous non-surfactant solutes) was demonstrated by the detection of pyrolysis radicals at 500-fold lower concentrations. Pyrolysis radicals were also found in the sonolysis of aqueous solutions containing only certain nitrone spin traps. The more hydrophobic the spin trap, the lower the concentration at which the pyrolysis radicals can be observed. The effect of varying the temperature of collapsing transient cavities in aqueous solutions of different rare gases and of N₂O on radical yields and on cell lysis of mammalian cells was investigated.     

Transformation of amino acid composition in bacterial cells of Lactobacillus bulgaricus during freeze-drying.

In the course of freeze-drying of a Lactobacillus bulgaricus strain, denaturation of protein leads to the separation of amino acids and their further susceptibility to deamination. This deamination of free and some bound amino acids is a secondary process and its intensity depends on the degree of denaturation, i.e., on protein and peptide degradation.A decarboxylation process takes place as well. The rate of decarboxylation is as high as the free amino acid pool is rich in acids.Based on these findings, it is reasonable to conclude that denaturation, deamination, and decarboxylation are biochemical processes which greatly influence the transformation of the native stereostructure of the complex, upon which the effect of freeze-drying on the survival level of cell suspension is dependent.     

E.s.r. of free radicals in aqueous solutions of substituted pyrimidines.

Reactions of OH radicals with methyl and ethyl derivatives of uracil, cytosine and thymine in aqueous solutions have been investigated. Photolysis of H2O2 was used to generate OH radicals and the radicals on the base derivatives were spin-trapped using t-nitrosobutane and identified with the help of e.s.r. spectroscopy. Addition of OH radicals was found to take place predominantly to the C(5)--C(6) double bond of the bases. H-abstraction from the methyl group occurred in the N(1) methyl derivatives of uracil, cytosine and thymine. Radicals formed by H-abstraction from the methyl group were also detected for 3-methyluracil, thymine, 1-methylthymine and 1-ethylthymine. Introduction of a methyl or ethyl group at the N(1) position of uracil, cytosine and thymine causes an increase in the C(6) proton coupling and a decrease in the N(1) splitting for radicals formed by OH addition at the C(5) position.     

Protein damage and degradation by oxygen radicals. II. Modification of amino acids

Exposure of proteins to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical (.OH + O2-) causes gross structural modification. Such modified proteins can undergo spontaneous fragmentation or can exhibit substantial increases in proteolytic susceptibility. In the present study, with the representative protein bovine serum albumin (BSA), we report that alterations to primary structure underlie such gross structural modifications. All amino acids in BSA were susceptible to modification by both .OH and .OH + O2- +O2), although tryptophan, tyrosine, histidine, and cysteine were particularly sensitive. At a radical/BSA molar ratio (nmol of radicals/nmol of BSA) of 10, we observed an average 9-10% destruction of amino acids; whereas at a ratio of 100, the average loss was 45%. Decreasing tryptophan fluorescence provided a useful index of amino acid loss and exhibited a clear dose dependence with .OH or with .OH + O2- (+O2). Linear production of the biphenol bityrosine was observed with .OH treatment. In contrast, .OH + O2- (+O2) induced only a limited bityrosine production rate which reached an early plateau. Studies with various chemical scavengers (t-butyl alcohol, isopropyl alcohol, mannitol, urate) and gasses (N2O, N2, O2, air) revealed that .OH is the primary radical responsible for all amino acid modifications, but that O2- and O2 can further transform the products of .OH reactions. Thus, O2-/O2 can potentiate .OH-dependent destruction of many amino acids (e.g. tryptophan) while inhibiting production of bityrosine by reacting with tyrosyl (phenoxyl) radicals. No amino acid loss or bityrosine production occurred with exposure to O2- (+O2) alone. Amino acid modifications caused both by .OH alone and by .OH + O2- (+O2) progressively affected the overall electrical charge of BSA. In a pH range of 3.7-6.2, some 16 new isoelectric focusing bands were induced by .OH, and some eight new bands were induced by .OH + O2- (+O2). The alterations to primary structure observed provide the key to an understanding of the link between oxidative modification and increased proteolytic susceptibility.     

Chemical transformations of proteinogenic amino acids during their sublimation in the presence of silica

Proteinogenic amino acids were sublimed in gram quantities in the presence of silica. High-performance liquid chromatographic and fast-atom-bombardment mass-spectrometric measurements showed that diketopiperazines (DKPs) were the main products of silica catalyzed thermal transformations in the case of aliphatic bifunctional amino acids. Intensive formation of products of decarboxylation, deamination and some other reactions was observed in the cases of trifunctional amino acids and phenylalanine. Possible mechanisms of reactions occured have been discussed. Principal possibility of gas-solid-phase DKP synthesis has been demonstrated for bifunctional aliphatic amino acids.     

PEPT1-mediated uptake of dipeptides enhances the intestinal absorption of amino acids via transport system b(0,+)

Free amino acids and short chain peptides are the main digestion products of dietary proteins in the small intestine. Whether there is a direct interference in transport of both groups of degradation products is not known. We used human intestinal Caco-2 cells to investigate whether the absorption of dipeptides by the peptide transporter PEPT1 alters the apical uptake of free cationic and neutral amino acids. Influx of L-[3H]Arg into Caco-2 cells was Na+-independent and mediated mainly by the b(0,+) system recognizing both cationic and neutral amino acids. Preincubation of cells with 10 mM of selected neutral, mono- or dicationic dipeptides increased the influx of L-Arg up to fourfold. Preloading with equivalent concentrations of the corresponding free amino acids also increased L-Arg influx but dipeptides always proved to be more efficient. The observed trans-stimulation was found to be specific for cationic amino acids since transport of L-[3H]Ala remained unaffected. We here demonstrate for the first time a direct interplay in amino acid and peptide transport in intestinal cells that may selectively alter the kinetics of amino acid absorption.     

Gamma-radiation-induced interactions between amino acids and glucagon

The interaction of glucagon and phenylalanine mediated by the OH . radical causes formation of higher molecular weight products of glucagon and phenylalanine, loss of amino acid residues in glucagon, and formation of adducts of glucagon and phenylalanine. The relative yields of these products depend upon the molar ratio of phenylalanine to glucagon in solution. At low ratios, glucagon aggregation and loss of amino acid residues predominate; at high ratios, the formation of phenylalanine dimers (and possible trimers and tetramers) predominates. The formation of adducts reaches a maximum at a phenylalanine:glucagon molar ratio of 3-4, and then decreases gradually, as the molar ratio increases, but is still discernible even at high molar ratios. Mechanisms for the formation of adducts are suggested. The influence of the primary aqueous radical intermediates, OH., H., and e-aq, on adduct formation has been evaluated for several different amino acids by irradiating in the presence of specific radical scavengers. For the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), OH. is considerably more effective than e-aq for mediating adduct formation, whereas for histidine and methionine, these primary radicals are equally effective.     

Interaction of amino acids with glycyl-glycine transport in the mammalian intestine

In order to investigate a possible interaction between free amino acids and dipeptides during their mucosal uptake in man and monkey, perfusion studiesin vivo and uptake studiesin vitro using labelled and non-labelled dipeptides and amino acids have been carried out. In contrast to the observations of other workers, inhibition of glycyl-glycine uptake was observed with free leucine and methioninc but not with glycine, proline, hydroxyproline or alanine. Leucine and methionine caused inhibition of cytosol glycyl-glycine hydrolase activity, while glycine had no effect. The dipeptide uptake and dipeptide hydrolysis by cytosol enzyme was competitively inhibited by leucine. Although brush border glycyl-glycine hydrolase was also inhibited by leucine, the inhibition was noncompetitive. These data indicate that a few free amino acids can interact with dipeptides during uptake. This interaction might occur either at the transport step or at the stage of intracellular dipeptide hydrolysis. The work reported here was carried out at Wellcome Research Unit, Christian Medical College and Hospital, Vellore 632 004.     

E.S.R. of spin-trapped radicals in aqueous solutions of 5-halo derivatives of nucleic acid constituents: reactions of hydrated electrons, hydroxyl radicals and U.V. photolysis

In order to obtain information concerning the mechanism of radio- and photosensitization due to 5-halogen substituted nucleic acid constituents, the free radicals produced in iodo-, bromo-, chloro- and fluoro-derivatives of uracil, uridine and deoxyuridine by reaction with hydrated electrons and with hydroxyl radicals and by direct U.V. photolysis have been studied by e.s.r. and spin-trapping. t-Nitrosobutane was used as the spin-trap. From 5-halogenated bases (except 5-fluorouracil) U.V. photolysis and reactions with hydrated electrons produced the uracilyl radical which was subsequently spin-trapped. When hydroxyl radical reactions were studied, the free radical at the N(1) position of the base was identified. From 5-fluorouracil U.V. photolysis generated the alpha-halo radical at the C(5) position of the base. For 5-halogenated ribonucleosides and deoxyribonucleosides, free radicals located on the sugar moiety were observed for reactions with hydrated electrons, hydroxyl radicals and for U.V. photolysis. The implications of these results for understanding the mechanism of radio- and photosensitization by 5-halogenated nucleic acids are discussed.     

E.s.r. of spin-trapped radicals in gamma-irradiated aqueous solutions of nucleic acids and their constituents.

The gamma-radiolysis of de-aerated neutral aqueous solutions of uracil, thymine, cytosine and of the corresponding nucleosides and nucleotides and of calf-thymus DNA was investigated. For uracil and thymine, the U.V. photolysis of aqueous solutions containing H2O2 was also studied. The short-lived radicals were spin-trapped by tert-nitrosobutane and identified by electron-spin-resonance spectroscopy. For all compounds two or more radicals were observed, and these could be distinguished by following the thermal decay of the spin adducts. Radicals formed by the addition of H or OH at the C(5) or C(6) positions of the pyrimidine derivatives were observed in all cases. Sodium formate was used as a scavenger for H and OH to identify the radicals formed by eaq-. Spin-trapped radicals in gamma-irradiated aqueous solutions of polynucleotides exhibited broad e.s.r. lines. For DNA gel, additional narrow lines due to scission products were also found.