Physico-chemical characterization of products of unfolding of cytochrome c by calcium chloride.
Cytochrome c (cyt c) denaturation by calcium chloride (CaCl2) and guanidine hydrochloride (GdnHCl) denaturation in presence of low fixed concentrations of CaCl2 has been carried out by UV/Vis spectrophotometry at pH 7.0 and 25 degrees C. The unfolding process was followed by measuring changes in difference molar extinction coefficient around 400 nm and delta epsilon 290. The products of denaturation were further characterized by intrinsic viscosity ([eta]) measurements. It has been observed that the reversible unfolding of cyt c by CaCl2 occurs in two distinct stages or involves three species (or states) namely N<-->X<-->D. Characterization of the native state, N intermediate state, X and the end product, D suggests that (i) During N<-->X only heme is exposed and no secondary structure unfolding occurs so that X state remains as compact as the native state. (ii) Stage X<-->D shows the melting of secondary structure. (iii) The end product corresponds to a random coil. and (iv) Thermodynamic characterization of the end product shows that heme plays an important role in the stability of the protein and removal of heme will lead to the unfolding of cyt c. Mixed denaturation shows a highly cooperative reversible transition between the native and denatured conformation. Analysis of the mixed results shows that (1) Gdn+ does not have any binding site (s) on the native cyt c, (2) there is one binding site for Ca2+ which stabilizes the protein, and (3) the binding constant, ks, is 5 M-1 for Ca2+.
Ahmad
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Z
Centre for Biosciences, Jamia Millia Islamia, New Delhi, India.
Ahmad
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F
D002122:Calcium Chloride / Q000494:pharmacology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003574:Cytochrome c Group / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013053:Spectrophotometry; D013816:Thermodynamics
D003574:Cytochrome c Group; D006146:Guanidines; D019791:Guanidine; D002122:Calcium Chloride