HELIX-COIL TRANSITION OF ISOLATED AMINO TERMINUS OF RIBONUCLEASE
HELIX-COIL TRANSITION OF ISOLATED AMINO TERMINUS OF RIBONUCLEASE
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DOI：
10.1021/bi00779a019
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发表时间：
1971-01-01
期刊：
影响因子：
2.9
通讯作者：
KLEE, WA
中科院分区：
文献类型：
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作者：
BROWN, JE;KLEE, WA
改进Gross和Witkop(1962)的溴化氰法制备多肽1-13(C-肽)。在一种典型的制剂中，将504毫克的牛胰腺核糖核酸酶A(Sigma XII A)添加到50.4毫升的70%甲酸中，其中含有1.25克溴化氰(伊士曼有机化学品，白色标签)。该溶液与核糖核酸酶蛋氨酸残基的摩尔比为1：80，在密闭烧瓶中于室温下缓慢搅拌24小时。然后反应混合物用5-6体积的无离子水稀释，壳冷冻，冻干。C-肽通过Sephadex G-25(粗)柱，118×6.5 cm，0.2 m醋酸为溶剂，分两次从C‘-蛋白中分离出来。用相同的溶剂通过1×30 cm Sephadex G-10柱，可实现一些轻微的进一步纯化。由Alan Neims博士使用Spackman等人(1958)的自动化程序为我们进行的冷冻干燥产物的氨基酸分析给出了以下结果，以及括号中的理论值：赖氨酸2.17(2)，组氨酸0.98(1)，精氨酸0.83(1)，苏氨酸0.70(1)，谷氨酸3.10(3)，丙氨酸3.10(3)，苯丙氨酸0.85(1)，天冬氨酸0.03(0)，丝氨酸0.01(0)，甘氨酸0.01(0)，高丝氨酸+高丝氨酸内酯0.52(1)。在色谱图上没有观察到其他峰。产物在pH 8.5下的纸层析显示两个吲哚三酮阳性斑点，在0.1n NaOH中短暂暴露时，其中较快的斑点转化为较慢的(较弱的碱性)斑点。Park等人(1963)将这种行为归因于残基13以高丝氨酸及其内酯的平衡混合物的形式存在(Armstrong，1949)。暴露量
Materials and MethodsPeptide 1-13 (C-peptide) was prepared by a modification of the cyanogen bromide procedure of Gross and Witkop (1962). In a typical preparation, 504 mg of bovine pancreatic ribonuclease A (Sigma Type XII A) was added to 50.4 ml of 70% formic acid containing 1.25 g of cyanogen bromide (Eastman Organic Chemicals, White Label). This solution, which has a molar ratio of ribonuclease methionine residues: CNBr of 1: 80, was stirred slowly in a closed flask at room temperature for 24 hr. The reaction mixture was then diluted with 5-6 volumes of ion-free water, shell frozen, and lyoph-ilized. C-peptide was separated from C'-protein by two passages through a Sephadex G-25 (coarse) column, 118 X 6.5 cm, using 0.2 m acetic acid as solvent. Some slight further purification could be effected by a passage through a 1 X 30 cm Sephadex G-10 column using the same solvent. Amino acid analysis of the lyophilized product, kindly performed for us by Dr. Alan Neims using the automated procedure of Spackman et al.(1958), gave the following results, with the theoretical values in parenthesis: lysine 2.17 (2), histidine 0.98 (1), arginine 0.83 (1), threonine 0.70 (1), glutamic acid 3.10 (3), alanine 3.10 (3), phenylalanine 0.85 (1), aspartic acid 0.03 (0), serine 0.01 (0), glycine 0.01 (0), and homoserine plus homoserine lactone 0.52 (1). No other peaks were observed on the chromatograms. Paper electrophoresis of the product at pH 8.5 shows two ninhydrin-positive spots, the faster of which is converted into the slower (less basic) on brief exposure of the peptide to 0.1 n NaOH. Parks et al.(1963) have ascribed this behaviorof the peptide to the fact that residue 13 exists as an equilibrium mixture of homoserineand its lactone (Armstrong, 1949). Exposure