Showing papers by "Charuvila T. Aravindakumar published in 2002"

Kinetics of the anaerobic reaction of nitric oxide with cysteine, glutathione and cysteine-containing proteins: implications for in vivo S-nitrosation

Abstract: A study is made of the kinetics of the anaerobic reaction of nitric oxide (˙NO) with cysteine and glutathione in relation to its potential physiological importance for the S-nitrosation of cysteine-containing peptides and proteins. The kinetics of the reaction with cysteine (the basic reagent unit) is studied most extensively and it is found that the rate constant is directly proportional to the degree of ionization (α), k = 0.37 × 103α M−1 s−1, offering clear proof for a mechanism based on electrophilic attack of ˙NO on thiolate anions. The rate constant for glutathione is considerably lower than for cysteine at identical pH, which can be attributed to its higher pKa; steric effects do not appear to affect the reactivity of glutathione significantly. On the basis of the rate equations obtained and of similar data for bovine serum albumin and metallothionein-1 a number of calculations were performed with the aim of determining the relative importance of the reaction of ˙NO with O2vs. the direct reaction of ˙NO with peptide and protein thiols under in vivo conditions. The results clearly show that in cells that as a rule contain an appreciable concentration of glutathione the autoxidation of ˙NO and thus the reaction of higher oxides of nitrogen (˙NO2, N2O3) with thiol groups in peptides and proteins does not play a role of any significance with respect to the formation of S-nitrosothiols, as the direct reaction of ˙NO with the thiolate group in glutathione leading to the formation of GS–˙NO− is much faster than the reaction of ˙NO with O2. The difference in reactivity is less pronounced in the case of bovine serum albumin, but again the electrophilic attack clearly is more important than the autoxidation at most physiological ˙NO concentrations. Direct electrophilic attack of ˙NO on metallothionein is of no practical significance, as the process is very much slower than the attack on glutathione.

Properties of the OH Adducts of Hydroxy-, Methyl-, Methoxy-, and Amino-Substituted Pyrimidines: Their Dehydration Reactions and End-Product Analysis

Abstract: Reactions of hydroxyl radicals (•OH) with 2-amino-4-methyl pyrimidine (AMP), 2-amino-4,6-dimethyl pyrimidine (ADMP), 2-amino-4-methoxy-6-methyl pyrimidine (AMMP), 2-amino-4-hydroxy-6-methyl pyrimidine (AHMP), 4,6-dihydroxy-2-methyl pyrimidine (DHMP), 2,4-dimethyl-6-hydroxy pyrimidine (DMHP), 6-methyl uracil (MU), and 5,6-dimethyl uracil (DMU) have been studied by pulse radiolysis and steady-state radiolysis techniques at different pH values. The second-order rate constants of the reaction of •OH with these systems are of the order of (2−9) × 10^9 dm^3 mol^(-1) s^(-1) at near neutral pH. The difference in the spectral features of the intermediates at near neutral pH and at higher pH (10.4) obtained with these pyrimidines are attributed to the deprotonation of the OH adducts. The G(TMPD•+) obtained at pH ∼ 6, from the electron-transfer reactions of the oxidizing intermediates with the reductant, N,N,N‘,N‘-tetramethyl-p-phenylenediamine (TMPD), are in the range (0.2−0.9) × 10^(-7) mol J^(-1) which constituted about 3−16% oxidizing radicals. These yields were highly enhanced at pH 10.5 in the case of AHMP, DHMP, DMU, and MU (G(TMPD^(•+)) = 3.8−5.5 ≅ 66−95% oxidizing radical). On the basis of these results, it is proposed that a nonoxidizing C(6)-ylC(5)OH radical adduct is initially formed at pH 6 which is responsible for the observed transient spectra. The high yield of TMPD•+ at higher pH is explained in terms of a base-catalyzed conversion (via a dehydration reaction) of the initially formed C(6)-ylC(5)OH adduct (nonoxidizing) to C(5)-ylC(6)OH adduct which is oxidizing in nature. Among the selected pyrimidines, such a dehydration reaction was observed only with those having a keto (or hydroxy) group at the C(4) position of the pyrimidine ring. Qualitative analyses of the products resulting from the OH adducts of DHMP (at pH 4.5) and DMHP (at pH 6) were carried out using HPLC-ES-MS and a variety of products have been identified. Glycolic and dimeric products were observed as the major end-products. The product profiles of both DHMP and DMHP have shown that the precursors of the products are mainly the C(6)-ylC(5)OH and the H adduct radicals. The identified products are formed mainly by disproportionation and dimerization reactions of these radicals. The mechanistic aspects are discussed.

Reaction of oxide radical ion (O .– ) with substituted pyrimidines

Abstract: Pulse radiolysis technique has been used to investigate the reaction of oxide radical ion (O.–) with 4,6-dihydroxy-2-methyl pyrimidine (DHMP), 2,4-dimethyl-6-hydroxy pyrimidine (DMHP), 5,6-dimethyl uracil (DMU) and 6-methyl uracil (MU) in strongly alkaline medium. The second-order rate constants for the reaction of O.– with these compounds are in the range 2-5 × 108 dm3 mol–1 s–1. The transient absorption spectra obtained with DHMP have two maxima at 290 and 370 nm and with DMHP have maxima at 310 and 470 nm. The transient spectrum from DMU is characterized by its absorption maxima at 310 and 520 nm and that of MU by its single maximum at 425 nm. The intermediate species were found to react with N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) with high G(TMPD.+) values ranged between 3.9 × 10–7 molJ–1 and 4.8 × 10–7 molJ–1. These radicals undergo decay by second-order kinetics (2k/∈ = 1.0-1.7 × 106 s–1). The reaction of O.– with the selected pyrimidines is proposed to proceed through a hydrogen abstraction from the methyl group forming allyl type radicals. These are mainly oxidizing radicals and hence readily undergo electron transfer reactions with TMPD.