Homer K A, Beighton D
Hunterian Dental Research Unit, London Hospital Medical College, United Kingdom.
Anal Biochem. 1990 Nov 15;191(1):133-7. doi: 10.1016/0003-2697(90)90399-t.
Intact fluorescein isothiocyanate-labeled proteins have relatively low background fluorescence at excitation and emission wavelengths of 495 and 525 nm, respectively. Degradation of these substrates leads to exposure of covalently linked fluorescein isothiocyanate molecules and to a concomitant increase in relative fluorescence at these wavelengths. The increase in relative fluorescence is proportional to the degree of protein degradation. This phenomenon provides the basis for a sensitive assay for bacterial protease activity. There is no requirement for the removal of undegraded substrate from the assay mixture prior to the measurement of fluorescence. Assays can be performed in 96-well microtiter trays, enabling a large number of samples and their respective controls to be processed simultaneously and repeated determinations of fluorescence values may be made on the same assay.
完整的异硫氰酸荧光素标记蛋白在激发波长495nm和发射波长525nm时具有相对较低的背景荧光。这些底物的降解会导致共价连接的异硫氰酸荧光素分子暴露，并使这些波长下的相对荧光随之增加。相对荧光的增加与蛋白质降解程度成正比。这一现象为细菌蛋白酶活性的灵敏检测提供了基础。在测量荧光之前，无需从检测混合物中去除未降解的底物。检测可在96孔微量滴定板中进行，能够同时处理大量样品及其各自的对照，并且可以对同一检测重复测定荧光值。
