Stabilization of Cytochrome C Reductase using Osmolytes

Abstract

In the present investigation, we have used cytochrome c reductase (cyt-c) to understand its unfolding process and also to study the protective ability of osmolytes on the unfolding process of cyt- c reductase. Our study mainly aims at understanding the stabilization of cyt-c reductase since its plays a major role in mitochondrial electron transport chain as well as in the apoptosis of the cell. It catalyzes the conversion of cyt-c oxidized form to reduced form. Cyt- c reductase was derived from breast cancer cell line MDA MB 231 and its unfolding process was studied thermally and in the presence of chemical denaturants like urea and guanidium hydrochloride (GdnHCl). The study was also carried out in presence of various osmolytes like glycerol, dimethysulfoxide (DMSO), sucrose and trehalose. Salt like MgCl2 was also added to the cell lysate to observe the effect of the conformational state of cyt- c reductase. The extent of unfolding of the protein was expressed in terms of the biological activity retained by the enzyme through measuring the change in the absorbance at 550nm due to the conversion of cy-c oxidized form to reduced form. Results indicated that under the application of heat the enzyme started unfolding at 40◦C and the enzyme showed an excess biological activity (150% ) till 50◦C and then the activity decreased sharply and dropped to zero at 60◦C indicating the complete denaturation of the enzyme. During thermal unfolding of the enzyme, 0.5M glycerol showed 100% activity at 60◦C whereas trehalose protected at 0.75M and 1M concentration and helped cyt-c reductase to retain approximately 75-85% activity. At 80◦C the enzyme activity was lost completely and the osmolytes also did not show any protection. During unfolding of cyt-c reductase by urea, glycerol at 0.72M showed 102% activity at 0.5M urea concentration and at 1M urea concentration, sucrose at 0.25M, 0.5M retained 114% and 106% activity. Sucrose showed protective effect only at 0.25M and 0.5M urea concentrations. Tehalose and DMSO in the concentrations 0.25M, 0.5M, 0.75M, and 1M showed a protective effect against denaturation by urea whereas the increasing concentrations of other osmolytes had a little protective effect on activity of cyt-c reductase. During unfolding of cyt-c reductase by GdnHCl, at higher concentrations of glycerol like at 4M, the enzyme was not denatured at 2M and rather 20% enzyme activity was retained. Trehalose at 0.25M, 0.5M.0.75M and 1M concentrations showed an enhanced activity from 20% to 40% at 2M GdnHCl concentration and maximum protection by trehalose was observed at 0.125M GdnHCl concentration nearly up to 90%. Sucrose had shown protective effect only at 1M concentration of GdnHCl. At 1M, 2M concentrations of GdnHCl, 0.75M, 1M DMSO showed highest protection nearly by 80%. DMSO is showing maximum protection at 2M concentration of GdnHCl where no other osmolytes had this much protection as DMSO. Salt like MgCl2 were also used to observe its stabilizing effect on the activity of cyt-c reductase.