[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 substrate Abz-Pro-Arg-Gly-Nph (I), whose action is based on intramolecular 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 the intramolecular fluorescence energy transfer. In substrate (I), a 4-nitrophenylhydrazide group was first used as an acceptor of excitation energy of the 2-aminobenzoyl group. The substrate is poorly hydrolyzed by thrombin (kcat/K(m) = 1.4 x 10(3) M-1 s-1) and is efficiently cleaved by trypsin (kcat/K(m) = 3.15 x 10(6) M-1 s-1). The hydrolysis of (II) can be monitored both spectrophotometrically, by absorbance at 405 nm, and from the increase in fluorescence at 340 nm. In the efficiency of hydrolysis with thrombin (kcat/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 designing substrates with the intramolecular fluorescence energy transfer for a variety of proteolytic enzymes.
Kholodovich
V V
VV
Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kiev, Ukraine.
Kara
D I
DI
Gershkovich
A A
AA
Kibirev
V K
VK
Karabut
L V
LV
Klimenko
I V
IV
Korneliuk
A I
AI
D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry*; D010659:Phenylhydrazines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry
C113950:2-aminobenzoylprolyl-arginyl-glycyl-4-nitrophenylhydrazine; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010659:Phenylhydrazines; C113951:tryptophyl-prolyl-arginyl-4-nitroaniline; D014364:Tryptophan; D014357:Trypsin; D013917:Thrombin
Novye donorno-aktseptornye pary dlia fluorogennykh substratov s vnutrimolekuliarnym perenosom énergii fluorestsetsii dlia trombina i tripsina.