Kandori Kazuhiko, Uoya Yumi, Ishikawa Tatsuo
School of Chemistry, Osaka University of Education, Asahigaoka 4-698-1, Kashiwara, Osaka 582-8582, Japan.
J Colloid Interface Sci. 2002 Aug 15;252(2):269-75. doi: 10.1006/jcis.2002.8495.
The objective of this study was to examine the effects of acetonitrile (AN) on the adsorption behavior of bovine serum albumin (BSA) onto calcium hydroxyapatite [Ca10(PO4)6(OH)2 Ca10, Hap] materials by combining the ultraviolet (UV) and circular dichroism (CD) measurements of BSA solution. The structural change of BSA molecules with addition of AN was investigated by UV and CD spectroscopy measurements prior to studying adsorption behavior of BSA onto Hap. The CD spectra revealed that the fraction of alpha-helical content of BSA is remarkably decreased at AN concentrations above 30 vol.%, while beta-sheet content is increased. On the other hand, the percentages of random coil and turn contents were decreased only slightly. In addition to this secondary structural change of BSA, the UV spectra suggested that the tertiary structure of protein molecules was also changed by the addition of large amounts of AN; BSA molecules associate to form molecular aggregates at [AN]> or =40 vol.%. From the adsorption of BSA onto Hap particles (ca. 30 nm in the particle length) from a water-AN mixed solution, it was revealed that the adsorption behavior of BSA strongly depends on the change of secondary and tertiary structures of BSA by addition of AN. The contraction of BSA molecules at low AN concentrations (10-20 vol.%) gave their small cross-sectional area, providing a large amount of adsorption (n(BSA)), although n(BSA) was decreased above 30 vol.% AN by enlargement of BSA molecules with solvation and unfolding some alpha-helix domains. The n(BSA) values of the systems with AN exhibited a maximum; n(BSA) was increased at a lower BSA concentration region, although it was decreased at a higher BSA concentration due to self-association. Accompanying the change of n(BSA) with AN addition, the maxima of electrophoretic mobility (em) of the Hap particles were observed for the systems with AN, although the em of Hap particles was normally increased and saturated with increase in protein coverage for the native structure on the system without AN. On the other hand, because the aggregated BSA molecules could be cooperatively bound, the adsorption of BSA onto the Hap particles with large size (108 nm in the particle length) was enhanced in the presence of AN.
本研究的目的是通过结合牛血清白蛋白（BSA）溶液的紫外（UV）和圆二色性（CD）测量，研究乙腈（AN）对牛血清白蛋白在羟基磷灰石[Ca10(PO4)6(OH)2，Ca10，Hap]材料上吸附行为的影响。在研究BSA在Hap上的吸附行为之前，通过UV和CD光谱测量研究了添加AN后BSA分子的结构变化。CD光谱显示，当AN浓度高于30体积%时，BSA的α-螺旋含量分数显著降低，而β-折叠含量增加。另一方面，无规卷曲和转角含量的百分比仅略有下降。除了BSA的这种二级结构变化外，UV光谱表明，添加大量AN也会改变蛋白质分子的三级结构；当[AN]≥40体积%时，BSA分子缔合形成分子聚集体。通过从水-AN混合溶液中将BSA吸附到Hap颗粒（颗粒长度约为30 nm）上，发现BSA的吸附行为强烈依赖于添加AN后BSA二级和三级结构的变化。在低AN浓度（10 - 20体积%）下，BSA分子的收缩使其横截面积变小，从而提供大量的吸附量（n(BSA)），尽管在AN浓度高于30体积%时，由于溶剂化导致BSA分子增大并展开一些α-螺旋结构域，n(BSA)会降低。含AN体系的n(BSA)值呈现最大值；在较低的BSA浓度区域n(BSA)增加，尽管在较高的BSA浓度下由于自缔合n(BSA)会降低。随着添加AN时n(BSA)的变化，含AN体系中观察到Hap颗粒电泳迁移率（em）的最大值，尽管对于不含AN的体系，Hap颗粒的em通常会随着蛋白质覆盖度的增加而增加并达到饱和。另一方面，由于聚集的BSA分子可以协同结合，在存在AN的情况下，BSA在大尺寸（颗粒长度为108 nm）的Hap颗粒上的吸附增强。
