Isoelectric focusing purity criteria and 1H NMR detectable spectroscopic heterogeneity in the major isolated monomer hemoglobins from Glycera dibranchiata.
Isoelectric focusing purity criteria and 1H NMR detectable spectroscopic heterogeneity in the major isolated monomer hemoglobins from Glycera dibranchiata.
复制标题
来自 Glycera dibranchiata 的主要分离单体血红蛋白的等电聚焦纯度标准和 1H NMR 可检测的光谱异质性。
DOI：
10.1021/bi00398a037
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Satterlee,JD
中科院分区：
文献类型：
--
作者：
Constantinidis,I;Satterlee,JD
新墨西哥州大学化学系，阿尔伯克基，新墨西哥州87131接收1986年11月6日;修订的Mandarin pt接收1987年6月18日摘要：三种主要的单体血红蛋白已经从二鳃单囊藻的红细胞中分离出来。它们对血红素蛋白的结构-功能研究的重要性在于它们已被证明具有特殊的氨基酸取代。在这些蛋白质中，E-7位置被亮氨酸占据，而不是更常见的远端组氨酸。与肌红蛋白相比，这种取代改变了血红素配体结合环境的极性。因此，G。二鳃类单体血红蛋白引起了人们的广泛关注。然而，到目前为止，还没有制定纯度标准。在这里，我们证明，对于所有的甘油单体血红蛋白，多个线图案显示在高压等电聚焦（IEF）凝胶。大多数这些线被证明是血红素相关现象的结果，可以理解的基础上的氧化和连接状态的血红素铁的变化。多线模式未表明单体血红蛋白制备物中存在显著杂质。马心肌红蛋白也表现出类似的行为。当聚焦单独的脱辅基蛋白凝胶时，IEF凝胶上的多条线图案消失。在这种情况下，除组分II外，所有单体血红蛋白均出现单一条带，组分II显示两条条带，一条主要条带和一条次要条带。次要带被认为是一种修饰的载脂蛋白形式。它对聚焦前的脱辅基蛋白处理敏感，并取决于IEF凝胶是否预聚焦。从这个分析中，IEF被证明是一个有价值的纯度标准，我们的单体血红蛋白组分II制剂的纯度为97%一个珠蛋白。建立单体组分II血红蛋白的纯度对于解释该组分的甲（水）形式的超精细质子NMR位移模式是重要的。NMR结果表明，组分II也存在两种类型的光谱异质性，并且这些异质性与蛋白质纯度无关。其中之一是占血红素异构，或位置逆转的血红素口袋里。第二个原因是由于珠蛋白II一级序列中CD 1位的苯丙氨酸环占据了多个位置。二鳃海鞘（Glycera dibranchiata）是一种海洋环节动物，在有核红细胞中具有血红蛋白。这些细胞的总血红蛋白含量可通过凝胶过滤分离成聚合物和单体形式（Vinogradov等人，1970），并且单体部分可进一步分解成五种血红蛋白（Kandler & Satterlee，1983; Kandler等人，1984; Cooke & Wright，1985 a）。该单体血红蛋白级分的重要性在于蛋白质从其结晶的事实，该蛋白质既可以测序（Imamura等人，1972）和晶体学（Padlan & Love，1974）一致认为，组氨酸E-7（远端组氨酸）被亮氨酸的特殊替代。考虑到
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131 Received November 6, 1986; Revised Manuscript Received June 18, 1987 abstract: Three major monomeric hemoglobins have been isolated from the erythrocytes of Glycera dibranchiata. Their importance to structure-function studies of heme proteins lies in the fact that they have been shown to possess an exceptional amino acid substitution. In these proteins, the E-7position is occupied by leucine rather than the more common distal histidine. This substitution alters the polarity of the heme ligand binding environment compared to myoglobin. Due to this, the G. dibranchiata monomer hemoglobins are attracting much attention. However, until now no purity criterion has been developed. Here we demonstrate that, for all of the Glycera monomer hemoglobins, multiple line patterns are shown on high-voltage isoelectric focusing (IEF) gels. Most of these lines are shown to be a consequence of heme-related phenomena and can be understood on the basis of changes in oxidation and ligation state of the heme iron. The multiple line pattern does not indicate significant impurities in the monomer hemoglobin preparations. Similar behavior is also demonstrated for horse heart myoglobin. The multiple line patterns on IEF gels disappear when gels of the apoproteins alone are focused. Single bands occur in this case for all of the monomer hemoglobins except component II, which displaystwo bands, one major and one minor. The minor band is found to be a modified apoprotein form. It is sensitive to apoprotein handlingprior to focusing and depends upon whether the IEF gel is prefocused or not. From this analysis, IEF is shown to be a valuable purity criterion, and the purity of our monomer hemoglobin component II preparation is 97% one globin. Establishing the purity of the monomer component II hemoglobin is important for interpreting the hyperfine proton NMR shift pattern of the met (aquo) form of this component. The NMR results show that two types of spectroscopic heterogeneity are also presentin component II, and these are unrelated to the protein purity. One of these is accounted for by heme isomerism, or positional reversal within the heme pocket. The second is due to the adoption of multiple positions by a phenylalanine ring at position CD1 in the globin II primary sequence.Glycera dibranchiata, a marine annelid, possesses hemoglobin in nucleated erythrocytes. The total hemoglobin content of these cells is separable into polymeric and monomeric forms by gel filtration (Vinogradov et al, 1970), and the monomer fraction is further resolvable into five hemoglobins (Kandler & Satterlee, 1983; Kandler et al., 1984; Cooke & Wright, 1985a). The importance of this monomer hemoglobin fraction resides in the fact that a protein was crystallized from it which both sequencing (Imamura et al., 1972) and crystallography (Padlan & Love, 1974) agree has the exceptional replacement of histidine E-7 (distal histidine) by leucine. In view of the