Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase

Abstract

Oxidation is one of the major pathways for denaturation of proteins during storage, and also a potential problem in protein production, isolation and purification processes. In this study, a number of additives have been tested for their protective effects against oxidation in the presence of metal ions and hydrogen peroxide. Porcine muscle lactate dehydrogenase (LDH) was used as a model protein. Oxidation and denaturation of the enzyme were followed as activity loss, modification of certain amino acids, altered secondary structure and aggregation. Loss of activity during metal-catalysed oxidation was accompanied by structural damage of the enzyme, which was not the case during oxidation with peroxide. The best protectant during both modes of oxidation was found to be the polycation, poly(ethyleneimine) (PEI), followed by EDTA. Both chemicals also increased the enzyme's half-life during oxidation with a mixture of copper ions and hydrogen peroxide. Ammonium sulphate was an effective stabilizer during metal-catalysed oxidation, while sorbitol, sucrose and hydroxyectoine provided moderate stabilization. The ectoine was also stabilizing against oxidation with hydrogen peroxide, as was poly(ethylene glycol), whereas sorbitol enhanced the rate of enzyme inactivation. The stability of LDH towards denaturation by both oxidation and temperature was increased by addition of both PEI and sorbitol, as indicated by the melting-temperature profiles of the enzyme.