Domain-Specific Stabilization of Structural and Dynamic Responses of Human Serum Albumin by Sucrose

Abstract

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>