马心脏细胞色素的全合成c.(1-66)片段的制备与表征。

C Di Bello, L Gozzini
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引用次数: 0

摘要

采用自动固相分步法在PAM树脂上合成了马心脏细胞色素c的天然(1-66)序列。采用定量茚三酮和Edman降解等分析方法对合成过程进行了监测。经HF裂解后,采用半制备离子交换和RP-HPLC相结合的方法纯化肽段。通过HPLC、氨基酸组成、电泳、抗体结合、胰蛋白酶和凝乳胰蛋白酶定位等不同标准来确定纯化合成肽的均匀性。在对Acm-Cys残基进行去保护和CNBr切割Met65- glu66肽键,同时将Met65残基转化为活化的c -末端[Hse65]内酯后,该纯化的合成肽已与synthetic(66-104)联合用于构象辅助连接实验,以产生完全合成的[Hse65]apocytochrome c。给出了一种与天然马心脏全细胞色素c相对应的功能分子。
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Total synthesis of horse heart cytochrome c. Preparation and characterization of the (1-66)apofragment.

A peptide corresponding to the native (1-66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid-phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion-exchange and RP-HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino-acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm-Cys residues and CNBr cleavage of the Met65-Glu66 peptide bond with simultaneous transformation of the Met65 residue into the activated C-terminal [Hse65]lactone, this purified synthetic peptide has been utilized for conformation-assisted joining experiments in combination with synthetic (66-104) to produce fully synthetic [Hse65]apocytochrome c. This latter, after mitochondria-mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.

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