Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.
The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005452:Fluoresceins / Q000138:chemical synthesis*; D007700:Kinetics; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011789:Quantum Theory; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D005452:Fluoresceins; C034357:fluorescein diester; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator