Spectroscopic Studies on the Conformational Stability of Hemocyanin of Pila virens (Lamarck, 1822) in the Presence of Temperature and Detergents

Abstract

Gastropods are endowed with a soluble copper containing, blue coloured respiratory protein known as hemocyanin. In the present study, for the first time, hemocyanin from Pila virens, a fresh water gastropod endemic to Indian sub-continent was studied for its conformational stability in the presence of temperature and various detergents by different spectroscopic techniques viz. UV–Vis, fluorescence and circular dichroism spectroscopy. Results from UV–Vis spectroscopy revealed that, absorbance corresponding to active site moieties were decreased with increase in temperature from 30 to 70 °C. Whereas ‘thermal reversibility’ studies showed the increase in absorbance of active site with decrease in temperature from 70 to 20 °C (is an indicative of the characteristic feature of hemocyanins—that oxygen is reversibly bound to the copper in the active site). Results from CD spectroscopy revealed that the temperature-induced conformational changes on the secondary structure of hemocyanin was reversible up to 70 °C. Beyond 80 °C, the protein was completely denatured and assumed an increased random coil conformation. Hemocyanin in the presence of high concentration of detergents lost its structure as evident from UV–Vis fluorescence (evident from the red shifts in the emission maxima accompanied by quenching in emission intensities) and CD spectroscopy. Stability of active site moieties in the presence of different detergents was also studied by UV–Vis spectroscopy. The biophysical techniques gave insights into the varied effects of each of the different denaturants on the conformational stability of hemocyanin from Pila virens.