Mechanism of acid-induced unfolding of Bovine serum albumin: Probing the unfolding intermediates by the scanning HPLC/SAXS/UV-vis system
Yi-Qi Yeh1*, Kuei-Fen Liao1, Wei-Ru Wu1, Chun-Jen Su1, Po-Chang Lin1, Ying-Jen Shiu1, Meng-Ciao Ho2, U-Ser Jeng1,3
1X-ray scattering group, National Synchrotron Radiation Research Center, Hsin-Chu, Taiwan
2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
3Department of Chemical Engineering, National Tsing Hua University, Hsin-Chu, Taiwan
* presenting author:Yi-Qi Yeh,
Serum albumin, the most abundant protein in vertebrate blood plasma, has a good binding capacity for fatty acids, hormones, commonly used drug, water, and essential metal ions. Bovine serum albumin (BSA, 583 amino acids, MW 66.5kDa), a serum albumin protein derived from cows, has long been widely used in immunodiagnostics. The BSA crystal structure is resolved, however, only very recently. Nevertheless, correlations of the solution structures with functions of the protein are still difficult to be elucidated, owing to the complicate and coexisting polymorphic states of monomers, dimers, and oligomers.
In this study, we have developed an on-line high-performanc liquid chrotomography (HPLC) system at 23A small- and wide-angle X-ray scattering (SAXS/WAXS) endstation of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. Further incorporated with an UV-Vis absorption spectrometer at the sample area, the HPLC/SAXS/UV-vis system allows separating the coexisting BSA monomer, dimers, and oligomers along the elute sample flow path for simultaneous SAXS/UV-Vis mesaurements, under a thermodsated sample temperature range of 288-337K and sample solution pH values between 2.1 to 7.2. The SAXS results reveal that the folding-unfolding behaviors of BSA monomers exhibit multistep unfolding process, with at least three intermediate structures. The intermediate structures are extracted from the SAXS data, using GASBOR protocol provided by the EMBO group. The intermediate structures are correlated to the protein crystal structure for a possible unfolding sequence of BSA. Implication of the intermediate unfolding steps is further addressed in terms of the thermodynamic parameters extracted from the SAXS data based on a modified Ising model for the unfolding free energy of each local unfolding group of BSA. Since albumin undergoes reversible conformational modification in pH 2-7 [3], the pH selectively control on BSA may help undersatnding in ligand/drug release and distribution mechanism.

Keywords: protein infolding, HPLC/SAXS, Bovine serum albumin