SPECTROSCOPIC STUDY OF HEMIN - HUMAN SERUM-ALBUMIN SYSTEM
SPECTROSCOPIC STUDY OF HEMIN - HUMAN SERUM-ALBUMIN SYSTEM
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DOI：
10.1111/j.1432-1033.1974.tb03295.x
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发表时间：
1974-01-01
期刊：
影响因子：
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通讯作者：
GRATZER, WB
中科院分区：
文献类型：
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作者：
BEAVEN, GH;CHEN, SH;GRATZER, WB
已重新检查了氯化血红素与人血清白蛋白的相互作用。结合血红素的吸收光谱与溶液中未络合的单体血红素的吸收光谱相同，并且基于白蛋白与无铁原卟啉IX的相互作用，表明铁不参与与蛋白质的相互作用。可以识别氰化铁衍生物和高铁血红蛋白的亚铁血红素衍生物，但不能识别叠氮化物或氟化物衍生物。结合血红素引起外在的棉花效应，这是不同的详细的衍生物，并在复杂的原卟啉IX分光光度滴定表明，有一个强结合位点的血红素和几个较弱的网站。后者与强络合物相反的旋光性有关。的结合配置文件是受pH值在很宽的范围内，由离子强度或由40%（v/v）二甲基亚砜/水的存在下，其中游离血红素是单体的影响不大。用分光光度法、圆二色谱法和荧光法研究了血红素与白蛋白的结合。血红素的结合淬灭蛋白质荧光，其特征逐渐从色氨酸型变为酪氨酸型。因此，初级结合位点的血红素强烈地特异性淬灭色氨酸。根据在一系列蛋白质浓度下的荧光滴定，构建了结合等温线，并给出了50 μ M − 1的强位点缔合常数。从结合等温线的基础上吸收测量，我们可以推断存在至少四个网站具有低得多的结合常数。发现长链脂肪酸阴离子仅与血红素竞争较弱的结合位点，因此可以基本消除每摩尔蛋白质超过1摩尔的结合。开链四吡咯，胆红素，被发现，与早期的工作一致，不与氯化血红素竞争，就任何配体的最强结合位点而言。然而，在较弱的站点之间，获得了竞争的证据。
The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron‐free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 μM−1. From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long‐chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open‐chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.