QUALITATIVE DETERMINATION OF N-TERMINAL AMINO-ACIDS OF PEPTIDES AND PROTEINS WITH COBALT(III) CHELATES
QUALITATIVE DETERMINATION OF N-TERMINAL AMINO-ACIDS OF PEPTIDES AND PROTEINS WITH COBALT(III) CHELATES
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DOI：
10.1042/bj1350507
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发表时间：
1973-01-01
影响因子：
4.1
通讯作者：
CREASER, EH
中科院分区：
文献类型：
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作者：
BENTLEY, KW;CREASER, EH
1.本文报道了用β-羟基水合三乙烯四胺钴（Ⅲ）离子β-[Co（trien）（OH）（OH_2）]~（2+）水解N-末端肽键的方法。该方法已被证明与22个小肽和10个蛋白质。2.该方法是快速的（一个N-末端氨基酸测定可以很容易地在一天内完成），它不涉及酸水解步骤，因此不破坏不稳定的氨基酸，它涉及使用容易制备的廉价试剂。3.释放的N-末端氨基酸可以被鉴定为它们的钴（III）衍生物，或直接鉴定为氨基酸或鉴定为它们的丹磺酰化衍生物。4.该方法是将1 μmol肽或蛋白质与β-[Co（trien）（OH）（OH 2）]2+试剂在pH8.0、45°C下反应3 h。加入0.5M磷酸盐缓冲液（pH10.5），在45 ℃下反应10 min，可使N端双齿氨基酸-钴配合物裂解，产物可直接鉴定。对于10 nmol肽的更高灵敏度，通过处理（用NaCN（0.1 m，40°C，30 min）或H2S或NaBH 4（25°C，5 min））从复合物制备游离氨基酸，干燥，丹磺酰化，并通过高压电泳鉴定丹磺酰-氨基酸。该方法不受4- 8 m-尿素的影响，但不会裂解封闭的N-末端酸。
1. A method ofN-terminal peptide-bond hydrolysis with thecis-β-hydroxyaquo(triethylenetetramine)cobalt(III) ion, i.e. β-[Co(trien)(OH)(OH2)]2+, is reported. The method has been demonstrated with 22 small peptides and ten proteins. 2. The procedure is rapid (anN-terminal amino acid determination can be made easily in one day), it involves no acid hydrolysis step and thus no destruction of labile amino acids, and it involves the use of easily prepared inexpensive reagents. 3. The releasedN-terminal amino acids can be identified as their cobalt(III) derivatives, or directly as the amino acid or as their dansylated derivatives. 4. The method is to treat 1 μmol of peptide or protein with β-[Co(trien)(OH)(OH2)]2+reagent at pH8.0, 45°C for 3h. Addition of 0.5m-phosphate buffer, pH10.5 at 45°C for 10min cleaves theN-terminal bidentate amino acid–cobalt complex, which can be identified directly. For greater sensitivity with 10nmol of peptide) the free amino acid is prepared from the complex by treatment (with NaCN (0.1m, 40°C, 30min), or H2S or NaBH4(25°C, 5min), dried, dansylated and the dansyl-amino acid identified by high-voltage electrophoresis. The method is unaffected by the presence of 4–8m-urea, but will not cleave blockedN-terminal acids.