STUDIES ON HUMAN LACTOFERRIN BY ELECTRON-PARAMAGNETIC RESONANCE, FLUORESCENCE, AND RESONANCE RAMAN-SPECTROSCOPY
STUDIES ON HUMAN LACTOFERRIN BY ELECTRON-PARAMAGNETIC RESONANCE, FLUORESCENCE, AND RESONANCE RAMAN-SPECTROSCOPY
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DOI：
10.1021/bi00558a026
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发表时间：
1980-01-01
期刊：
影响因子：
2.9
通讯作者：
LOEHR, TM
中科院分区：
文献类型：
--
作者：
AINSCOUGH, EW;BRODIE, AM;LOEHR, TM
通过对金属取代的人乳铁蛋白的荧光、共振拉曼光谱和EPR光谱的研究，证实了乳铁蛋白与血清转铁蛋白的相似性。与铁转铁蛋白和铜转铁蛋白的情况一样，脱脂乳铁蛋白S的本征荧光显著猝灭是由于铁、铜、铬、锰和钴与特定的金属结合部位相互作用所致。这些金属-乳铁蛋白的激光激发产生了.apprx的共振拉曼光谱特征。1605、1505、1275和1175厘米-1。这些带的特征是酪氨酸与金属离子的配位作用，正如先前对血清转铁蛋白观察到的那样，并允许每个金属-乳铁蛋白中的主吸收带(最大吸收峰在400-465 nm之间)被分配给金属离子和酪氨酸配体之间的电荷转移。与血清转铁蛋白一样，乳铁蛋白中的两个金属结合部位可以通过EPR谱区分，特别是与铬(III)取代的蛋白质。在乳铁蛋白的2个位置中，只有1个位置允许Fe(III)取代Cr(III)。乳铁蛋白与血清转铁蛋白的不同之处在于它对铁的亲和力增强。动力学研究表明，脱脂乳铁蛋白吸收Fe(III)的速度是脱铁蛋白的10倍。更显著的构象变化发生在金属与乳铁蛋白结合时，证实了在锰(III)-乳铁蛋白中产生了额外的EPR可检测到的铜(II)结合位点。从乳铁蛋白中去除铁所需的较低pH会导致蛋白质发生永久性变化，这可以通过铁(III)吸收速率的变化和在铜(II)存在下的EPR谱的变化来判断。通过广泛透析柠檬酸(PH 2)来生产脱乳铁蛋白的常见方法显然对蛋白质有不利影响。
Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman and EPR spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin''s intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III) and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at .apprx. 1605, 1505, 1275 and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as was observed previously for serum transferrins and permit the principal absorption band (.lambda.max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. As in serum transferrin the 2 metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only 1 of the 2 sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin differs from serum transferrin in its enhanced affinity for Fe. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)-citrate is 10 times faster for apolactoferrin than for apotransferrin. The more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for Fe removal from lactoferrin causes permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). The common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) apparently has an adverse effect on the protein.