NEW DONOR-ACCEPTOR PAIRS FOR FLUOROGENIC SUBSTRATES WITH INTRAMOLECULAR FLUORESCENCE ENERGY-TRANSFER FOR THROMBIN AND TRYPSIN

New substrates for thrombin and trypsin are described: a fluorogenic s ubstrate Abz-Pro-Arg-Gly-Nph (I), whose action is based on intramolecu lar fluorescence energy transfer, and H-D-Trp-Pro-Arg-pNA (II), which can be used both as a chromogenic substrate and as a substrate with th e intramolecular fluorescence energy transfer. In substrate (I), a 4-n itrophenylhydrazide group was first used as an acceptor of excitation energy of the 2-aminobenzoyl group. The substrate is poorly hydrolyzed by thrombin (k(cat)/K-m = 1.4 x 10(3) M-1 s(-1)) and is efficiently c leaved by trypsin (k(cat)/K-m = 3.15 x 10(6) M-1 s(-1)). The hydrolysi s of (II) can be monitored both spectrophotometrically, by absorbance at 405 nm, and from the increase in fluorescence at 340 nm. In the eff iciency of hydrolysis with thrombin (k(cat)/K-m = 3.0 x 10(6) M-1 s(-1 )), compound (II) is comparable with the known chromogenic substrates for this enzyme. The proposed donor-acceptor pairs are promising in de signing substrates with the intramolecular fluorescence energy transfe r for a variety of proteolytic enzymes.