A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50-300-fold. The physical basis for this increase in sensitivity is discussed.
Research Article| February 01 1983
Rhodamine-based compounds as fluorogenic substrates for serine proteinases
Biochem J (1983) 209 (2): 299–307.
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S P Leytus, L L Melhado, W F Mangel; Rhodamine-based compounds as fluorogenic substrates for serine proteinases. Biochem J 1 February 1983; 209 (2): 299–307. doi: https://doi.org/10.1042/bj2090299
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