Corin Storkey

Reevaluation of the rate constants for the reaction of hypochlorous acid (HOCl) with cysteine, methionine, and peptide derivatives using a new competition kinetic approach.

Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H2O2 and Cl(-), Br(-), or SCN(-) to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5 × 10(8)M(-1)s(-1)). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6 × 10(8), 2.9 × 10(7), and 1.24 × 10(8)M(-1)s(-1), respectively. Similar experiments with methionine derivatives yielded k values of 3.4 × 10(7)M(-1)s(-1) for Met and 1.7 × 10(8)M(-1)s(-1) for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.

Preventing protein oxidation with sugars: scavenging of hypohalous acids by 5-selenopyranose and 4-selenofuranose derivatives.

Heme peroxidases including myeloperoxidase (MPO) are released at sites of inflammation by activated leukocytes. MPO generates hypohalous acids (HOX, X = Cl, Br, SCN) from H(2)O(2); these oxidants are bactericidal and are key components of the inflammatory response. However, excessive, misplaced or mistimed production can result in host tissue damage, with this implicated in multiple inflammatory diseases. We report here methods for the conversion of simple monosaccharide sugars into selenium- and sulfur-containing species that may act as potent water-soluble scavengers of HOX. Competition kinetic studies show that the seleno species react with HOCl with rate constants in the range 0.8-1.0 × 10(8) M(-1) s(-1), only marginally slower than those for the most susceptible biological targets including the endogenous antioxidant, glutathione. The rate constants for the corresponding sulfur-sugars are considerably slower (1.4-1.9 × 10(6) M(-1) s(-1)). Rate constants for reaction of the seleno-sugars with HOBr are ~8 times lower than those for HOCl (1.0-1.5 × 10(7) M(-1) s(-1)). These values show little variation with differing sugar structures. Reaction with HOSCN is slower (~10(2) M(-1) s(-1)). The seleno-sugars decreased the extent of HOCl-mediated oxidation of Met, His, Trp, Lys, and Tyr residues, and 3-chlorotyrosine formation, on both isolated bovine serum albumin and human plasma proteins, at concentrations as low as 50 μM. These studies demonstrate that novel selenium (and to a lesser extent, sulfur) derivatives of monosaccharides could be potent modulators of peroxidase-mediated damage at sites of acute and chronic inflammation, and in multiple human pathologies.

Mechanisms of degradation of the natural high-potency sweetener (2R,4R)-monatin in mock beverage solutions.

The sodium, potassium, or mixed sodium/potassium salt of the naturally occurring high-potency sweetener (2R,4R)-monatin, also known by the common name arruva, degrades over time in model beverage solutions in the presence of light. By use of UHPLC, LC-MS/MS, and peroxide assays, it has been demonstrated that degradation is accelerated by UV/visible light and the presence of trace metal ions. Data are presented that are consistent with a role for singlet oxygen (¹O₂), free radicals, and peroxides (both H₂O₂ and organic peroxides) in monatin oxidation. Separation of degradation products by UHPLC/HPLC or LC-MS/MS provided evidence for the formation of hydroxylated and peroxide species formed on the indole ring (mass increases 16 and 32, respectively) as well as multiple ring and side-chain oxidation and scission products. Model oxidation systems using the photosensitizer Rose Bengal as a source of ¹O₂ support the proposed photodegradation pathways.

Prevention of degradation of the natural high potency sweetener (2R,4R)-monatin in mock beverage solutions

Exposure of the naturally-occurring sweetener monatin to light and metal ions results in loss of both parent monatin and total indole (monatin plus monatin lactone/lactam) in mock beverage solutions, with an accompanying decrease in sweetness. In this study potential protective strategies to prevent degradation were investigated. Metal ion chelating resin, or the chelators EDTA and desferrioxamine decreased monatin and indole loss for solutions kept either in darkness or exposed to light. Tannic acid and Chinese bayberry extract both afforded protection, but this did not arise from a light filtering effect. Plastics with defined absorbance characteristics provided protection with this being wavelength dependent; yellow transparent PET plastic was most effective. The contribution of these interventions (metal ion removal/binding; antioxidant; light absorption) was additive, with combinations providing the greatest protective effect against monatin and indole loss. These results indicate that it is possible to minimise monatin degradation by appropriate choices of treatments, additives and container.