Unfolding pathway of cytochromec oxidase induced by ionic surfactants: Circular dichroism and picosecond time-resolved fluorescence studies

Abstract

The unfolding of the membrane protein, cytochrome c oxidase (CcO) induced by ionic surfaetants have been studied by using circular dichroism, optical absorbance and time resolved tryptophan fluorescence spectroscopic methods. Ionic surfactant cetyltrimethyl ammonium bromide (CTAB) was found to cause denaturation of this membrane protein leading to release of both, the heme a residues from CcO indicated by both CD and optical titration. Upon dissociation of the hemes from the protein matrix; the tryptophan fluorescence intensity of CcO increased drastically and the fluorescence lifetimes became much longer compared to the short lifetimes observed in the native protein. The shortest lifetime of 70 ps observed in the native protein due to strong quenching (energy transfer) of the beme groups, increased ~ 10-fold in the CTAB-unfolded protein indicating complete removal of the heme groups from the protein matrix. Remarkable differences were observed between the mode of actions of ionic surfactants and the commonly used denaturant guanidine hydrochloride. Improved data analysis of maximum entropy method showed that the lifetime distribution pattern in the two cases of unfolding were very different. The lifetimes in guanidine hydrochloride unfolded CcO were much shorter and more widely distributed indicating that the hemes are probably not separated away from the protein matrix and that the unfolded state is highly heterogeneous. Our results further showed that the lauryl maltoside inhibits denaturation of CcO by the ionic surfactant and the initial step of the denaturation possibly involves quantitative replacement of the lauryl maltoside by the ionic surfactant at the surface of the enzyme.

Kinetics of Structural Transitions Induced by Sodium Dodecyl Sulfate in α-Chymotrypsin

“…The concentration of the stock SDS solution was 320 mM. Using this solution, SDS parent solutions (16,32,48,64, and 80 mM) were prepared. This gives a range of final SDS concentrations when mixed with the chymotrypsin solution from 8 to 40 mM, which is a range similar to that used in Takeda's work on αand δ-chymotrypsin.…”

“…Kinetic investigations of structural transitions in proteins caused by SDS or other surfactants have also been undertaken by other research groups. ,− …”