Chang J Y
Biochem J. 1981 Dec 1;199(3):557-64. doi: 10.1042/bj1990557.
This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.
本文描述了一种用于多肽N端序列分析的手动方法，其灵敏度可达亚纳摩尔水平。采用二甲基氨基偶氮苯异硫氰酸酯/苯异硫氰酸酯双偶联法逐步降解多肽，通过反相高压液相色谱法鉴定释放出的氨基酸二甲基氨基偶氮苯乙内酰脲。二甲基氨基偶氮苯乙内酰脲是有色化合物，可在可见光区域检测，灵敏度极限为1皮摩尔（信噪比为5）。开发了一种高压液相色谱法，用于全面分析所有氨基酸二甲基氨基偶氮苯乙内酰脲衍生物，包括丝氨酸和苏氨酸的副产物。因此，无需使用自动测序仪或放射性材料，使用少于1纳摩尔的材料就有可能确定肽的完整序列和蛋白质的N端序列。
