CONFORMATIONAL-CHANGES AT THE ACTIVE-SITE OF BOVINE PANCREATIC RNASE-A AT LOW CONCENTRATIONS OF GUANIDINE-HYDROCHLORIDE PROBED BY PYRIDOXAL5'-PHOSPHATE

The alpha-amino group of Lys-1 and the epsilon-amino groups of Lys-41 and Lys-7 were labeled with pyridoxal 5'-phosphate (PLP) separately. T he effects of GdnHCl on the activities and the fluorescence properties of the derivatives were compared. Both the fluorescence intensity and anisotropy of the probe introduced at the active site Lys-41 decrease d obviously during denaturation by low concentrations of GdnHCl indica ting conformational change of the active site is a parallel event to t he inactivation of the enzyme. Time-correlated fluorescence lifetime m easurements revealed the existence of two conformational states of PLP -modified RNase A and a shift of the conformation in favor of the slow -decay component with increasing GdnHCl concentration. The decrease in activity of RNase A at low concentrations of GdnHCl is therefore due to partial unfolding of the molecule, particularly at the active site. The experimental results of this work support the suggestion that the conformation at the active site is more easily perturbed and hence mo re flexible than the molecule as a whole.