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

In the present work, we have investigated the denaturing and renaturing effects of urea and sucrose respectively, on the overall structure and on two different domains (domain-I and domain-III) of multi-domain human serum albumin (HSA). From circular dichroism measurements it could be seen that, the α-helicity of the overall structure, which is 65 % in its native state, decreases to 11 % in presence of 9 M urea and further increases to 25 % with the addition of 1 M sucrose. In order to study the domain-specific responses towards these external agents, HSA was covalently tagged with fluorescent probes N-(7-dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) and p-nitrophenyl coumarin ester (NPCE) at Cys-34 of domain-I and Tyr-411 of domain-III, respectively. The domain-wise unfolding/folding studies of HSA by steady state fluorescence spectroscopy reveal that domain-III is more labile to denaturation while the renaturating effect of sucrose is more pronounced on domain-I. The calculation of free energy change during denaturation due to urea divulges the presence of an intermediate state, which gets stabilized in the presence of sucrose. The renaturing or stabilizing effect of sucrose is attributed to the stabilization of this intermediate. From solvation dynamics studies it could be seen that the solvation dynamics near binding site of DACIA inside domain-I becomes faster in presence of urea and it becomes slower in presence of sucrose. However, in the case of NPCE-tagged domain-III, the effect of sucrose on solvation time is evident only at high concentrations of urea and the denaturing effect of urea is evident only at very low or zero concentration of sucrose.