Desfosses B, Cittanova N, Urbach W, Waks M
Unité de Formation et de Recherches Biomédicale des Saints Pères, Université René Descartes, Paris, France.
Eur J Biochem. 1991 Jul 1;199(1):79-87. doi: 10.1111/j.1432-1033.1991.tb16094.x.
The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Ka approximately 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-411 does not seem substantially modified by the 15% decrease affecting the alpha helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels.
已比较了人血清白蛋白在三种化学性质不同的配体（氧苯基布他松、丹磺酰肌氨酸和血红素）存在下，于缓冲液以及异辛烷、水与双（2-乙基己基）磺基琥珀酸钠或十六烷基三甲基溴化铵形成的反胶束中的行为，这些体系旨在模拟膜-水界面。胶束包合后，丹磺酰肌氨酸-白蛋白复合物发生解离，荧光发射光谱（从485nm红移至570nm）和荧光偏振测量结果均证实了这一点。相比之下，根据408nm处的Soret吸收带以及8.4×10⁴M⁻¹cm⁻¹的摩尔吸收系数判断，血红素-白蛋白复合物在反胶束中保持稳定。氧苯基布他松与白蛋白的结合曲线表明，虽然缔合常数保持不变（Ka约为1.0×10⁵M⁻¹），但只有一部分存在的白蛋白分子与配体发生反应。结果不受表面活性剂的性质和浓度影响。这些发现可根据大的胶束内表面积在人血清白蛋白中诱导的构象变化来解释。荧光发射最大值从缓冲液中的344nm蓝移至双（2-乙基己基）磺基琥珀酸钠胶束中的327nm，以及荧光团对N-溴代琥珀酰亚胺氧化的反应性/可及性降低，表明唯一的色氨酸-214微环境受到了扰动。然而，吲哚残基与酪氨酸-411之间的距离似乎并未因白蛋白分子α螺旋15%的减少而发生实质性改变。本文报道的结果表明，白蛋白与类似膜的界面相互作用产生了两个在膜表面和配体亲和力上不同的蛋白质亚群。因此，源于这种表面诱导效应的整体和局部构象变化可能构成一种在细胞膜水平起作用的促进配体释放的机制。
