Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase

ANDERSSON MM,; BRECCIA JD,; HATTI-KAUL R,

BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY

Portland Press

Año: 2000 vol. 32 p. 145 - 153

0885-4513

Oxidationis one of the major pathways for denaturationof proteins during storage, and also a potential problemin protein production, isolation and purification processes.In this study, a number of additives have beentested for their protective effects against oxidation inthe presence of metal ions and hydrogen peroxide.Porcine muscle lactate dehydrogenase(LDH) was usedas a model protein. Oxidation and denaturation of theenzyme were followed as activity loss, modification ofcertain amino acids, altered secondary structure andaggregation. Loss of activity during metal-catalysedoxidationwas accompanied by structural damageof theenzyme, which was not the case during oxidation withperoxide. The best protectant during both modes ofoxidation was found to be the polycation, poly-(ethyleneimine) (PEI), followed by EDTA. Bothchemicals also increased the enzyme?s half-life duringoxidation with a mixture of copper ions and hydrogenperoxide. Ammonium sulphate was an effectivestabilizer during metal-catalysed oxidation, while sorbitol,sucrose and hydroxyectoine provided moderatestabilization. The ectoine was also stabilizing againstoxidation with hydrogen peroxide, as was poly(ethyleneglycol), whereas sorbitol enhanced the rate of enzymeinactivation. The stability of LDH towards denaturationby both oxidation and temperature was increased byaddition of both PEI and sorbitol, as indicated by themelting-temperature profiles of the enzyme.