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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.