title: Does recovery in the spectral characteristics of GdnHCl-denatured Bacillus licheniformis α-amylase due to added calcium point towards protein stabilization?
abstract: 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 CaCl(2) in the incubation mixture produced significant recovery in spectral signals, being complete in presence of 10 mM CaCl(2), 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 CaCl(2) suggested significant aggregation of the protein in the absence of CaCl(2) 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 presence of aggregation.
authors.affiliation: Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, Muala Lumpur, Malaysia.
authors.forename: Adyani Azizah Abd
authors.initials: AA
authors.lastname: Halim
authors.forename: Shevin Rizal
authors.initials: SR
authors.lastname: Feroz
authors.forename: Saad
authors.initials: S
authors.lastname: Tayyab
mesh_terms: D001407:Bacillus / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002122:Calcium Chloride / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000516:alpha-Amylases; C501919:alpha-amylase, Bacillus licheniformis; D019791:Guanidine; D002122:Calcium Chloride