Use of domain specific ligands to study urea-induced unfolding of bovine serum albumin.
Urea-induced structural transitions in different domains of bovine serum albumin (BSA) were studied fluorometrically using domain specific ligands; chloroform, bilirubin, and diazepam. Urea denaturation of BSA showed a two-step, three-state transition with the accumulation of an intermediate around 4.8-5.2 M urea. During first transition (0-5.0 M urea), a continuous decrease (starting from 1.0 M urea) in diazepam (a ligand for domain III) binding and a late (from 3.0 M urea onward) decrease in chloroform (a ligand primarily for domain I) binding suggested major conformational changes in domain III and partial but significant loss of native conformation in domain I prior to intermediate formation. Absence of any decrease in bilirubin (a ligand for domain II) binding up to 4.5 M urea indicated non-involvement of domain II in the unfolding of BSA in this region. However, decrease in bilirubin binding during second transition reflected the unfolding of domain II and its separation from domain I.
Tayyab
S
S
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202002, India. btisamua@nde.vsnl.net.in
Sharma
N
N
Mushahid Khan
M
M
D000818:Animals; D001663:Bilirubin / Q000378:metabolism; D002417:Cattle; D002725:Chloroform / Q000378:metabolism; D003975:Diazepam / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000494:pharmacology*
D008024:Ligands; D012709:Serum Albumin; D002725:Chloroform; D014364:Tryptophan; D014508:Urea; D003975:Diazepam; D001663:Bilirubin