Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods.

Abstract:

: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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Flora K,Brennan JD,Baker GA,Doody MA,Bright FV

doi

10.1016/S0006-3495(98)77598-8

subject

Has Abstract

pub_date

1998-08-01 00:00:00

pages

1084-96

issue

2

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(98)77598-8

journal_volume

75

pub_type

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