Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods.
Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.
Department of Chemistry, Brock University, St. Catharines, Ontario L2S 3A1, Canada.
K
K
Flora
J D
JD
Brennan
G A
GA
Baker
M A
MA
Doody
F V
FV
Bright
D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D001665:Binding Sites; D003545:Cysteine; D004735:Energy Transfer; D005456:Fluorescent Dyes; D019791:Guanidine; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D012044:Regression Analysis; D012459:Salicylates; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D013997:Time Factors
D005456:Fluorescent Dyes; D012459:Salicylates; D012709:Serum Albumin; C038518:acrylodan; D015081:2-Naphthylamine; D019791:Guanidine; D003545:Cysteine