Interactive binding to the two principal ligand binding sites of human serum albumin: effect of the neutral-to-base transition

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• 作者：Yamasaki ; Keishi ; Maruyama ; Toru ; Yoshimoto ; Kaori ; Tsutsumi ; Yasuhiro ; Narazaki ; Ryuichi ; et. al.
• 关键词：Human serum albumin ; Ligand binding site ; Displacement study ; Allosteric interaction ; Neutral-to-base transition
• 刊名：Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular Enzymology
• 出版年：1999
• 出版时间：July 13, 1999
• 年：1999
• 卷：1432
• 期：2
• 页码：313-323
• 全文大小：313 K

文摘

The relationship between the two principal ligand binding sites, sites I and II, on human serum albumin (HSA) was quantitatively and qualitatively examined by equilibrium dialysis and fluorescence spectroscopy. Among the three subsite markers to site I, only the binding of dansyl-l-asparagine (DNSA), which is a subsite Ib marker (K. Yamasaki et al., Biochim. Biophys. Acta 1295 (1996) 147), was inhibited by the simultaneous binding of a site II ligand, such as ibuprofen and diazepam. This indicates that, in contrast to subsite Ib, subsites Ia and Ic do not strongly interact with site II. The thermodynamic characteristics for the coupling reaction between DNSA and ibuprofen and between DNSA and diazepam, which gave positive coupling free energies and negative values for both coupling enthalpy and entropy, indicated that the reaction process was entropically driven. Increase of pH from 6.5 to 8.2 caused an increase in coupling constant and entropy for the mutual antagonism between DNSA and the site II ligands on binding to HSA. The site II ligand-induced red-shift of λ_max and solvent accessibility of DNSA in subsite Ib were decreased when the albumin molecule was isomerized from the neutral (N) to the base (B) conformation in the physiological pH region. Based on these findings, we conclude that a ‘competitive’ like strong allosteric regulation exists for the binding of these two ligands to the N conformer, whereas for the B conformer this interaction can be classified as nearly ‘independent’. Since the distance between Trp-214, which resides within the site I subdomain, and Tyr-411, which is involved in site II, is increased by 6 Å during the N–B transition (N.G. Hagag et al., Fed. Proc. 41 (1982) 1189), we propose a mechanism for the pH-dependent antagonistic binding between subsite Ib and site II, which involves the transmission of ligand-induced allosteric effects from one site to another site, modified by changes in the spatial relationship of sites I and II caused by the N–B transition.