Stability of lactate dehydrogenase in metal-catalyzed oxidation solutions containing chelated metals

The role of metal chelators in altering the reactivity :of metal-catalyzed oxidation solutions containing iron or copper, ascorbate, and hydrogen peroxide was evaluated by measuring the loss of enzymatic activity of lactate dehydrogenase (LDH) and the formation of hydroxyl radicals. The chelators used were iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid, bicinchoninic acid, dihydrobenzoic acid, desferroxalamine mesylate, and phytate. All of the chelators tested at a 10:1 chelator: metal-ratio afforded some protection to LDH compared to the unchelated system; Selected chelators that afforded only partial protection to LDH were able to provide nearly complete protection at higher chelator:metal ratios. This protection is likely attributable to the chelators' ability to remove surface-bound metals and, thereby, reduce ''site-specific'' oxidation. Many metal-chelator pairs generated hydroxyl radicals. The rate and amount of hydroxyl radicals formed varied depending on the metal-chelator pair. Although large amounts of hydroxyl radicals did not always result in increased protein inactivation, the absence of radicals was required for complete protein protection. The chelator to metal ratio required to eliminate hydroxyl radical formation varied widely with the metal-chelator pair and was unusually high in some cases. The results suggest that metal-chelator pairs (of various ratios) can be screened for hydroxyl radical formation and that; those which: show no radical formation are expected to protect the protein. No definitive conclusions can be drawn concerning the stability of LDH in solutions capable of producing hydroxyl radicals as the rate and extent of hydroxyl radical formation was riot correlated simply to LDH inactivation under the conditions studied.