Does recovery in the spectral characteristics of GdnHCl-denatured Bacillus licheniformis α-amylase due to added calcium point towards protein stabilization?

Treatment of Bacillus licheniformis α-amylase (BLA) with guanidine hydrochloride (GdnHCl) produced both denatured and aggregated forms of the enzyme as studied by circular dichroism, fluorescence, UV difference spectroscopy, size exclusion chromatography (SEC), and enzymatic activity. The presence of CaCl2 in the incubation mixture produced significant recovery in spectral signals, being complete in presence of 10 mM CaCl2, as well as in enzymatic activity, which is indicative of protein stabilization. However, the SEC results obtained with GdnHCl-denatured BLA both in the absence and the presence of 10 mM CaCl2 suggested significant aggregation of the protein in the absence of CaCl2 and disaggregation in its presence. Although partial structural stabilization with significant retention of enzymatic activity was observed in the presence of calcium, it was far from the native state, as reflected by spectral probes. Hence, spectral results as to BLA stabilization should be treated with caution in the presen...