Equilibrium studies of the effect of difference in sequence homology on the mechanism of denaturation of bovine and horse cytochromes-c.
We have carried out equilibrium studies of the effect of the amino acid residue difference in the primary structure of bovine cytochrome-c (b-cyt-c) and horse cyt-c (h-cyt-c) on the mechanism of their folding <--> unfolding processes at pH 6.0 and 25 degrees C. It has been observed that guanidinium chloride (GdmCl)-induced denaturation of b-cyt-c follows a two-state mechanism and that of h-cyt-c is not a two-state process. This conclusion is reached from the coincidence and non-coincidence of GdmCl-induced transition curves of bovine and horse proteins, respectively, monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. These measurements on h-cyt-c in the presence of GdmCl in the concentration range 0.75-2.0 M also suggest that the protein retains all the native far-UV CD but has slightly perturbed tertiary interaction. The intermediate in the presence of these low denaturant concentrations does not have the structural characteristics of a molten globule as judged by the 8-Anilino-1-napthalene sulfonic acid (ANS) binding and near-UV CD experiments. We have also carried out thermal denaturation studies of bovine and horse cyts-c in the presence of GdmCl monitored by absorbance at 405 nm and far-UV CD at 222 nm. The heat-induced denaturation measurements in the presence of the denaturant show (1) that denaturation of b-cyt-c is a two-state process and that of h-cyt-c does not follow a two-state mechanism, and (2) that the enthalpy change on denaturation of both proteins strongly depends on GdmCl concentration.
Moza
Beenu
B
Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi-110 025, India.
Qureshi
Shabir H
SH
Ahmad
Faizan
F
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D019791:Guanidine / Q000494:pharmacology; D006736:Horses; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D019791:Guanidine