一种含有新型荧光氨基酸DL-2-氨基-3-(7-甲氧基-4-香豆基)丙酸的硫醚肽酶淬灭荧光底物。
A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid.
作者信息
Knight C G
机构信息
Department of Biochemistry, Strangeways Research Laboratory, Cambridge, U.K.
出版信息
Biochem J. 1991 Feb 15;274 ( Pt 1)(Pt 1):45-8. doi: 10.1042/bj2740045.
Abstract
DL-2-Amino-3-(7-methoxy-4-coumaryl)propionic acid, a new fluorescent amino acid (abbreviated to Amp), has been synthesized to provide an alternative to tryptophan in quenched fluorescent peptide substrates for peptidases. The model compound Ac-DL-Amp-NH2 was intensely fluorescent with an excitation maximum at 328 nm and an emission maximum at 392 nm. Fmoc (fluoren-9-ylmethoxycarbonyl)-DL-Amp was made to allow the solid-phase synthesis of Amp-containing peptides by the Fmoc-polyamide method. The peptide derivative Dnp (2,4-dinitrophenyl)-Pro-Leu-Gly-Pro-DL-Amp-D-Lys was cleaved by thimet peptidase at the Leu-Gly bond, with a 20-fold enhancement of fluorescence. The value of kcat./Km for thimet peptidase was 6.7 x 10(5) M-1.s-1, compared with the value of 2.4 x 10(5) M-1.s-1 for the tryptophan-containing analogue, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys.
摘要
DL-2-氨基-3-(7-甲氧基-4-香豆基)丙酸，一种新型荧光氨基酸（简称为Amp），已被合成出来，用于在肽酶的猝灭荧光肽底物中替代色氨酸。模型化合物Ac-DL-Amp-NH2具有强烈荧光，最大激发波长为328nm，最大发射波长为392nm。制备了芴甲氧羰基（Fmoc）-DL-Amp，以便通过Fmoc-聚酰胺法固相合成含Amp的肽。肽衍生物Dnp（2,4-二硝基苯基）-Pro-Leu-Gly-Pro-DL-Amp-D-Lys在Leu-Gly键处被硫醇肽酶裂解，荧光增强了20倍。硫醇肽酶的kcat./Km值为6.7×10(5)M-1·s-1，而含色氨酸类似物Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys的值为2.4×10(5)M-1·s-1。
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