Alkali-induced conformational transition in different domains of bovine serum albumin.
Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh--202002, India. rizwanhkhan@hotmail.com
Basir
B
Ahmad
Mohammad Zahid
MZ
Kamal
Rizwan Hasan
RH
Khan
D000468:Alkalies / Q000494:pharmacology*; D000818:Animals; D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002725:Chloroform / Q000737:chemistry; D003975:Diazepam / Q000737:chemistry; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism
D000468:Alkalies; D012710:Serum Albumin, Bovine; D002725:Chloroform; D003975:Diazepam; D001663:Bilirubin