Copper induced polymerization of hemoglobin from the ocean pout, Macrozoarces americanus.
Copper induced polymerization of hemoglobin from the ocean pout, Macrozoarces americanus.
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铜诱导来自美洲Macrozoarces americanus 的血红蛋白聚合。
DOI：
10.1086/bblv187n2p246
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Harrington,J
中科院分区：
文献类型：
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作者：
Borgese,TA;Bourke,S;Frias,B;Johnson,D;Harrington,J
血红蛋白在两栖动物、爬行动物和板鳃类中并不少见，但在硬骨鱼和人类中很少见。它们是研究蛋白质结构、功能和分子进化关系的有用模型。淡水硬骨鱼Hoplias malabarica（I），海洋硬骨鱼Lophius americanus（2）和小丑鱼Amphiprion（3）是唯一报道的具有血红蛋白的硬骨鱼。Hoplias和Lophius通过形成分子间二硫键来聚合，而小丑鱼的聚合机制至今仍不清楚。在人类中，血红蛋白Ta-Li、密西西比和Pot-to阿莱格雷（4，5，6）具有单一突变，涉及珠蛋白链上不同位点的半胱氨酸残基的取代。我们现在报告说，海洋pout有一个单一的血红蛋白与六个巯基基团每个四聚体和聚合，在体外，氧化后与铁氰化钾（K，Fe（CN），）或氯化铜（CuCl）。已经描述了通过DE-52纤维素色谱法纯化血红蛋白、通过G-100柱上的凝胶过滤法估计分子量以及用于珠蛋白亚基分析的尿素凝胶电泳的程序（2）。将血红蛋白与2至3倍摩尔过量的CuClZ在冰桶中孵育15分钟。通过透析或通过从用0.1M K-PO平衡的Sephadex G-25柱凝胶过滤除去过量的铜。缓冲液，pH 7.4。此后，将20-40 mg血红蛋白施加到G-100柱上并从G-100柱洗脱。收集1 ml级分，并在540 nm处测量各级分的吸光度。根据峰管的洗脱体积估计近似聚合物分子量。血红蛋白溶液（0.5- 2.0g/%）也通过每ml血红蛋白加入1 -3 mg铁氰化钾来氧化。将碘乙酰胺（IAA）-烷基化血红蛋白（15毫摩尔/毫克血红蛋白）透析过夜，离心，随后用CuCl 2处理。在另一个实验中，将60倍摩尔过量的β-巯基乙醇（B-ME）加入到CuCl*-处理的血红蛋白中，孵育1小时，透析3小时，然后进行G-100层析。未处理的血红蛋白和单独用CuClZ处理的血红蛋白用作对照，并透析相同的时间。
Hemoglobins that polymerize are not uncommon among amphibians, reptiles, and elasmobranchs, but are rare among teleosts and humans. They are useful models for the study of protein structure, function, and molecular evolutionary relationships. The freshwater teleost Hoplias malabarica (I), the marine teleost Lophius americanus (2), and the clown fish, Amphiprion (3) are the only bony fishes reported to have hemoglobins that polymerize. Hoplias and Lophius polymerize by forming intermolecular disulfide bonds, while the polymerization mechanism for the clown fish is, as yet, unknown. Among humans, hemoglobins Ta-Li, Mississippi, and Pot-to Alegre (4, 5, 6) have a single mutation involving the substitution of a cysteine residue at different loci on the globin chain. We now report that the ocean pout has a single hemoglobin with six sullhydryl groups per tetramer and polymerizes, in vitro, after oxidation with potassium ferricyanide (K, Fe (CN),) or cupric chloride (CuClJ. Procedures for hemoglobin purification by DE-52 cellulose chromatography, molecular weight estimations by gel filtration on G-100 columns, and urea gel electrophoresis for globin subunit analysis have been described (2). Hemoglobin was incubated with a two-to threefold molar excess of CuCIZ for 15 min in an ice bucket. The excess copper was removed by dialysis or by gel filtration from Sephadex G-25 columns equilibrated with 0.1 M K-PO., buffer, pH 7.4. Thereafter 20-40 mg of the hemoglobin was applied to and eluted from G-100 columns. One-ml fractions were collected and the absorbance of each measured at 540 nm. The approximate polymer molecular weight was estimated from the elution volume of the peak tube. Hemoglobin solutions (0.5-2.0 g/%) were also oxidized by adding l-3 mg of potassium ferricyanide per ml of hemoglobin. Iodoacetamide (IAA)-alkylated hemoglobin (15 rmoles/mg hemoglobin) was dialyzed overnight, centrifuged, and subsequently treated with CuC12.In a separate experiment, a 60-fold molar excess of beta-mercaptoethanol (B-ME) was added to CuCl*-treated hemoglobin, incubated for 1 h, and dialyzed for 3 h before G-100 chromatography. Untreated hemoglobin and hemoglobin treated with CuCIZ alone served as controls and were dialyzed for the same period.