皮摩尔水平的氨基酸分析。在多肽C末端序列分析中的应用。
Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.
作者信息
Chang J Y, Knecht R, Braun D G
出版信息
Biochem J. 1981 Dec 1;199(3):547-55. doi: 10.1042/bj1990547.
Abstract
Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.
摘要
用二甲基氨基偶氮苯磺酰氯标记的氨基酸可通过反相高压液相色谱法分离，并在可见光区域（436纳米）进行检测。通过使用由乙腈/水性缓冲液混合物组成的两种不同梯度系统，所有19种天然存在的氨基酸都可以在Zorbax ODS柱上分离。低至2 - 5皮摩尔的单个二甲基氨基偶氮苯磺酰基氨基酸能够可靠地进行定量分析，每次完整的氨基酸分析仅需要10 - 30纳克的二甲基氨基偶氮苯磺酰化蛋白质水解产物。这项新技术与任何当前涉及离子交换分离加荧光检测的氨基酸分析方法一样灵敏，并且在技术上要简单得多。通过将这种灵敏的氨基酸分析技术与羧肽酶相结合，我们已经能够在皮摩尔水平上确定多肽的C末端序列。
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