Evaluation of the extent of heterogeneity in the Glycera dibranchiata monomer haemoglobin fraction by the use of n.m.r. and ion-exchange chromatography.
Evaluation of the extent of heterogeneity in the Glycera dibranchiata monomer haemoglobin fraction by the use of n.m.r. and ion-exchange chromatography.
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使用 nmr 评估 Glycera dibranchiata 单体血红蛋白部分的异质性程度。
DOI：
10.1042/bj2260131
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Satterlee,JD
中科院分区：
文献类型：
--
作者：
Kandler,RL;Constantinidis,I;Satterlee,JD
已知 Glycera dibranchiata 的体腔血红蛋白可分离为单体和较高 Mr 级分。尽管相对于 Mr 表现出同质性，但单体部分的血红蛋白异质性程度从未得到充分测定。在本文中，我们证明无论分离过程中是否存在蛋白酶抑制剂，单体血红蛋白级分中都存在可重复检测的异质性。这些结果表明，考虑到与之前用于氨基酸测序、晶体学和动力学的制剂相同的时间尺度，单体血红蛋白部分是高度异质的。应用离子交换色谱和离子过滤方法从甘油单体血红蛋白组分中分离出四种可溶解的血红蛋白成分。其中三种成分的分离量足以使用质子核磁共振。作为区分个体血红蛋白的成功分析工具。这些结果并不令人意外。先前的几项研究表明单体部分的异质性较小。此外，甘油单体血红蛋白即使在相当低的分压下也能结合氧的能力归因于多种血红蛋白成分的功能多样性。目前的工作揭示了血红蛋白异质性的程度。结果表明，其范围比之前认为的更为广泛。对该单体部分的检查特别重要，因为晶体学表明单体部分的成分之一缺少 E-7（远端）组氨酸残基。因此，识别如此广泛的异质性对于许多先前发表的配体结合研究非常重要。
The coelomic haemoglobin of Glycera dibranchiata is known to be separable into monomeric and higher-Mr fractions. Although exhibiting homogeneity with respect to Mr, the extent of haemoglobin heterogeneity for the monomer fraction has never been adequately assayed. In the present paper we demonstrate that there exists in the monomer haemoglobin fraction reproducibly detectable heterogeneity regardless of the presence or absence of proteinase inhibitors during the isolations. These results show that, considered on the same time scale as previous preparations used for amino acid sequencing, crystallography and kinetics, the monomer haemoglobin fraction is highly heterogeneous. Application of ion-exchange chromatography and ion-filtration methods resulted in the isolation of four resolvable haem protein components from the Glycera monomer haemoglobin fraction. Three of these components were isolated in sufficient quantity to employ proton n.m.r. as a successful analytical tool for discriminating the individual haemoglobins. These results are not surprising. Several previous studies indicated less extensive heterogeneity in the monomer fraction. Moreover, the ability of the Glycera monomer haemoglobin to bind oxygen at even quite low partial pressures has been attributed to functional diversity originating in multiple haemoglobin components. The present work reveals the extent of the haemoglobin heterogeneity. The results show that it is more extensive than previously believed. Examination of this monomer fraction is particularly important, since crystallography indicates that one of the components of the monomer fraction lacks the E-7 (distal) histidine residue. As a consequence, the identification of such extensive heterogeneity is important to many previously published ligand-binding studies.