Peroxynitrite (ONOO⁻), a potent oxidant, mediates the oxidation of both nonprotein and protein sulfhydryls. It is formed when superoxide (O₂⁻) and nitric oxide (·NO) react, which can occur in endothelial cells, macrophages, and neutrophils. The reaction rates of peroxynitrite with free cysteine and the single thiol of albumin are significantly higher than those of hydrogen peroxide (H₂O₂), with second-order rate constants of 5,900 M⁻¹s⁻¹ and 2,600–2,800 M⁻¹s⁻¹, respectively. Unlike H₂O₂, which oxidizes thiolate anions, peroxynitrite preferentially reacts with the undissociated thiol form. It oxidizes cysteine to cystine and the thiol group of bovine serum albumin (BSA) to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid (ONOOH) is less effective than its anion, with oxidation likely mediated by decomposition products such as hydroxyl radicals and nitrogen dioxide. Peroxynitrite is highly reactive toward sulfhydryls, with a rate of reaction about 10³ times faster than H₂O₂ at 37°C and pH 7.4. This reaction may be a significant mechanism of oxygen radical-mediated toxicity. The study investigated peroxynitrite's reactions with protein and nonprotein sulfhydryl groups, comparing them with H₂O₂-mediated thiol oxidation. Sulfhydryls are critical targets for free radicals, with oxidation being a key mechanism of free radical-mediated toxicity. Thiols are essential for enzyme active sites and protein conformation. The study found that peroxynitrite oxidizes sulfhydryls more rapidly than H₂O₂, suggesting a role in oxygen radical toxicity. The study also examined the effects of metal chelators and pH on sulfhydryl oxidation. Desferrioxamine inhibited dimethyl sulfoxide and deoxyribose oxidation by peroxynitrous acid but had no effect on peroxynitrite-mediated sulfhydryl oxidation. Oxidation yields were higher at alkaline pH, with peroxynitrite oxidizing BSA and cysteine more efficiently. The pH dependence of the reaction rates indicated that peroxynitrite reacts preferentially with the undissociated thiol form. The pKa of BSA thiol was determined to be 7.86–8.00, while that of cysteine was 8.04. The study also compared the oxidation products of BSA and cysteine by peroxynitrite and H₂O₂. Peroxynitrite oxidizedPeroxynitrite (ONOO⁻), a potent oxidant, mediates the oxidation of both nonprotein and protein sulfhydryls. It is formed when superoxide (O₂⁻) and nitric oxide (·NO) react, which can occur in endothelial cells, macrophages, and neutrophils. The reaction rates of peroxynitrite with free cysteine and the single thiol of albumin are significantly higher than those of hydrogen peroxide (H₂O₂), with second-order rate constants of 5,900 M⁻¹s⁻¹ and 2,600–2,800 M⁻¹s⁻¹, respectively. Unlike H₂O₂, which oxidizes thiolate anions, peroxynitrite preferentially reacts with the undissociated thiol form. It oxidizes cysteine to cystine and the thiol group of bovine serum albumin (BSA) to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid (ONOOH) is less effective than its anion, with oxidation likely mediated by decomposition products such as hydroxyl radicals and nitrogen dioxide. Peroxynitrite is highly reactive toward sulfhydryls, with a rate of reaction about 10³ times faster than H₂O₂ at 37°C and pH 7.4. This reaction may be a significant mechanism of oxygen radical-mediated toxicity. The study investigated peroxynitrite's reactions with protein and nonprotein sulfhydryl groups, comparing them with H₂O₂-mediated thiol oxidation. Sulfhydryls are critical targets for free radicals, with oxidation being a key mechanism of free radical-mediated toxicity. Thiols are essential for enzyme active sites and protein conformation. The study found that peroxynitrite oxidizes sulfhydryls more rapidly than H₂O₂, suggesting a role in oxygen radical toxicity. The study also examined the effects of metal chelators and pH on sulfhydryl oxidation. Desferrioxamine inhibited dimethyl sulfoxide and deoxyribose oxidation by peroxynitrous acid but had no effect on peroxynitrite-mediated sulfhydryl oxidation. Oxidation yields were higher at alkaline pH, with peroxynitrite oxidizing BSA and cysteine more efficiently. The pH dependence of the reaction rates indicated that peroxynitrite reacts preferentially with the undissociated thiol form. The pKa of BSA thiol was determined to be 7.86–8.00, while that of cysteine was 8.04. The study also compared the oxidation products of BSA and cysteine by peroxynitrite and H₂O₂. Peroxynitrite oxidized