p-Aminobenzamidine as a fluorescent probe for the active site of serine proteases.
p-Aminobenzamidine is weakly fluorescent in neutral aqueous buffer, with excitation and emission maxima at 293 and 376 nm, respectively. Binding to trypsin results in a blue shift of the emission peak to 362 nm, and 50-fold fluorescence enhancement, while binding to thrombin causes a shift to 368 nm and a 230-fold fluorescence enhancement. This phenomenon is due to hydrophobic interactions, as evidenced by the similar properties observed when p-aminobenzamidine is dissolved in solvents of decreasing polarity. The absorbance spectrum of p-aminobenzamidine is red-shifted by formation of a complex with proteases, with the major difference peak appearing at 317 nm and 323 nm for trypsin and thrombin, respectively. The difference extinction coefficients were 6000 M-1 cm-1 for trypsin complex and 13,300 M-1 cm-1 for thrombin complex at the peak wavelengths. Titration of trypsin and thrombin with the probe indicated one binding site per molecule, with dissociation constants equal to the kinetically determined inhibition constants. The KD values for trypsin and thrombin were 6.1 and 65 microM, respectively. An important potential use of this probe is in studies of inhibitor and substrate binding by rapid reaction kinetic techniques. Using this probe to study the interaction of thrombin with antithrombin III yielded a bimolecular rate constant of 8.0 x 10(3) M-1 s-1, which compares favorably with the value of 8.7 x 10(3) M-1 s-1 obtained from discontinuous assays of the rate of thrombin neutralization.
D000578:Amidines / Q000494:pharmacology*; D001550:Benzamidines / Q000494:pharmacology*; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology*
D000578:Amidines; D001550:Benzamidines; D014361:Trypsin Inhibitors; C006500:4-aminobenzamidine; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013917:Thrombin