In vitro stability of various enzymes by proline from H2O2 mediated oxidative damage

Abstract

Plants under stress need to favour certain pathways so as to survive the stress period. Protection of specific enzymes by proline and other osmolytes could be one such mechanism to favour some pathways/processes. Therefore, the influence of osmolyte proline on conformational changes of various proteins caused by hydrogen peroxide (H₂O₂) was studied by intrinsic and extrinsic fluorescence emissions. H₂O₂ caused conformational change in proteins. Results indicated that for Alcohol dehydrogenase (AD) and Glutamate dehydrogenase (GD) enzymes, H₂O₂ induced conformational change was high and that for Glucose 6-phosphate dehydrogenase (G6PDH) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was low. Fluorescence and far-UV, CD measurements of catalase demonstrated that the H₂O₂ stabilized the protein secondary structure at low concentrations but destabilized it at higher concentrations. Intrinsic and ANS fluorescence results showed that proline at a concentration of 1.0 M prompted a reduction in the H₂O₂-induced exposed hydrophobic surfaces of studied enzymes, to different degrees which suggests its differential protective effect. Furthermore, SDS-PAGE studies revealed that proline was not able to reduce or inhibit the H₂O₂ mediated aggregation of GAPDH.