Detection and characterization using circular dichroism and fluorescence spectroscopy of a stable intermediate conformation formed in the denaturation of bovine carbonic anhydrase with guanidinium chloride

Abstract

Particularly stable elements of noncovalent structure in bovine carbonic anhydrase were detected and studied. These are present in a highly populated intermediate state formed during denaturation of the enzyme with guanidinium chloride. The intermediate was detected by analysis of the denaturation profiles and some of its structural properties were characterized by CD [circular dichroism] and fluorescence spectroscopy, including fluorescence polarization and lifetime measurements. Measurements were made on the Zn2+-enzyme, Co2+enzyme and apoenzyme to ascertain the structural effects of the active-site Zn2+. Kinetic measurements indicate that this intermediate is on the folding pathway from the random coil to the native state.