Rotenberg M, Margalit R
Biochem J. 1985 Jul 1;229(1):197-203. doi: 10.1042/bj2290197.
The binding equilibrium of deuteroporphyrin IX to human serum albumin and to bovine serum albumin was studied, by monitoring protein-induced changes in the porphyrin fluorescence and taking into consideration the self-aggregation of the porphyrin. To have control over the latter, the range of porphyrin concentrations was chosen to maker dimers (non-covalent) the dominant aggregate. Each protein was found to have one high-affinity site for deuteroporphyrin IX monomers, the magnitudes of the equilibrium binding constants (25 degrees C, neutral pH, phosphate-buffered saline) being 4.5 (+/- 1.5) X 10(7) M-1 and 1.7 (+/- 0.2) X 10(6) M-1 for human serum albumin and for bovine serum albumin respectively. Deuteroporphyrin IX dimers were found to bind directly to the protein, each protein binding one dimer, with high affinity. Two models are proposed for the protein-binding of porphyrin monomers and dimers in a porphyrin system having both species: a competitive model, where each protein molecule has only one binding site, which can be occupied by either a monomer or a dimer; a non-competitive model, where each protein molecule has two binding sites, one for monomers and one for dimers. On testing the fit of the data to the models, an argument can be made to favour the non-competitive model, the equilibrium binding constants of the dimers, for the non-competitive model (25 degrees C, neutral pH, phosphate-buffered saline), being: 8.0 (+/- 1.8) X 10(8) M-1 and 1.2 (+/- 0.6) X 10(7) M-1 for human serum albumin and bovine serum albumin respectively.
通过监测卟啉荧光中蛋白质诱导的变化，并考虑卟啉的自聚集现象，研究了去铁卟啉IX与人血清白蛋白和牛血清白蛋白的结合平衡。为了控制后者，选择卟啉浓度范围以使二聚体（非共价）成为主要聚集体。发现每种蛋白质对去铁卟啉IX单体都有一个高亲和力位点，在25℃、中性pH、磷酸盐缓冲盐溶液条件下，人血清白蛋白和牛血清白蛋白的平衡结合常数大小分别为4.5（±1.5）×10⁷ M⁻¹和1.7（±0.2）×10⁶ M⁻¹。发现去铁卟啉IX二聚体直接与蛋白质结合，每种蛋白质以高亲和力结合一个二聚体。对于同时存在这两种物质的卟啉系统中卟啉单体和二聚体与蛋白质的结合，提出了两种模型：一种竞争模型，其中每个蛋白质分子只有一个结合位点，可被单体或二聚体占据；一种非竞争模型，其中每个蛋白质分子有两个结合位点，一个用于单体，一个用于二聚体。在测试数据与模型的拟合度时，可以认为支持非竞争模型，在非竞争模型下（25℃、中性pH、磷酸盐缓冲盐溶液），二聚体的平衡结合常数分别为：人血清白蛋白8.0（±1.8）×10⁸ M⁻¹，牛血清白蛋白1.2（±0.6）×10⁷ M⁻¹。
