Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling.
Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling.
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通过片段偶联全合成S-氨基甲酰甲基牛脱细胞色素c。
DOI：
10.1111/j.1399-3011.1986.tb01810.x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Blake,J
中科院分区：
文献类型：
--
作者：
Blake,J
通过固相法合成了对应于104个氨基酸蛋白质牛脱辅基细胞色素c的完整序列的三个肽段。肽Ac-[Cys（Cam）14，17，GlyS 23]-脱辅基细胞色素c-（1-23）（I）、CF 3CO-[GlyS 60]-脱辅基细胞色素c-（24-60）（II）和CF 3CO-脱辅基细胞色素c-（61-104）（III）通过CM-纤维素层析、分配层析和/或HPLC纯化。使各肽与柠康酸酐反应以封闭所有赖氨酸侧链，并用10%肼处理61-104肽以除去三氟乙酰基，得到相应的肽Ia、IIa和IIIa。通过与硝酸银/N-羟基琥珀酰亚胺反应将肽IIa和IIIa偶联在一起，得到24-104序列。从氨基末端除去三氟乙酰基后，还偶联了肽Ia。用乙酸水溶液处理肽混合物除去柠康酰基，并通过CM-纤维素色谱法和HPLC纯化，得到0.6%产率的[Cys-（Cam）14，17]-脱辅基细胞色素c。合成产物经纸电泳、凝胶电泳、高效液相色谱、胰凝乳蛋白酶图谱和胰蛋白酶图谱鉴定，与天然牛细胞色素c样品一致。
Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid‐phase method. The peptides Ac‐[Cys(Cam)14,17, GlyS23]‐apocytochrome c‐(1–23) (I), CF3CO‐[GlyS60]‐apocytochrome c‐(24–60) (II), and CF3CO‐apocytochrome c‐(61–104) (III) were purified by chromatography on CM‐cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61–104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N‐hydroxysuccinimide to give the 24–104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM‐cellulose and HPLC gave a 0.6% yield of [Cys‐(Cam)14,17]‐apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.