Background:
Human Serum Albumin (HSA) is the most abundant protein present in
human blood plasma. It is a large multi-domain protein with 585 amino acid residues. Due to its
importance in human body, studies on the interaction of HSA with different external agent is of
vital interest. The denaturation and renaturation of HSA in presence of external agents are of particular
interest as they affect the biological activity of the protein.
Objective:
The objective of this work is to study the domain-specific and overall structural and
dynamical changes occurring to HSA in the presence of a denaturing agent, urea and a renaturing
agent, sucrose.
Methods:
In order to carry out the domain-specific studies, HSA has been tagged using N-(7-
dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) at Cys-34 of domain-I and pnitrophenyl
coumarin ester (NPCE) at Tyr-411 site in domain-III, separately. Steady-state absorption,
emission and solvation dynamic measurements have been carried out in order to monitor the
domain-specific alteration of HSA caused by the external agents. The overall structural change of
HSA have been monitored using circular dichroism spectroscopy.
Results:
The α-helicity of HSA was found to decrease from 65% to 11% in presence of urea and
was found to further increase to 25% when sucrose is added, manifesting the denaturing and
renaturing effects of urea and sucrose, respectively. The steady state studies show that domain-III is
more labile towards denaturation as compared to domain-I. The presence of an intermediate state is
observed during the denaturation process. The stabilization of this intermediate state in presence of
sucrose is attributed as the reason for the stabilization of HSA by sucrose. From solvation dynamics
studies, it could be seen that the solvation time of DACIA inside domain-I of HSA decreases and
increases regularly with increasing concentrations of urea and sucrose, respectively, while in the
case of NPCE-tagged domain-III, the effect of sucrose on solvation time is evident only at high
concentrations of urea.
Conclusion:
The denaturing and renaturing effects of urea and sucrose could be clearly seen from
the steady state studies and circular dichroism spectroscopy measurements. A regular change in
solvation time could only be observed in the case of domain-I and not in domain-III. The results
indicate that the renaturing effect of sucrose on domain-III is not very evident when protein is in its
native state, but is evident in when the protein is denatured.</P>