4,4'-Bis[8-(phenylamino)naphthalene-1-sulfonate] binding to human thrombins: a sensitive exo site fluorescent affinity probe.
The binding of the fluorescent probe 4,4'-bis[8-(phenylamino)naphthalene-1-sulfonate] (bis-ANS) to human alpha- and gamma-thrombins was investigated. Bis-ANS binds in a 1:1 complex to both forms of the enzyme, with Kd = 14.8 +/- 2.2 microM and 5.8 +/- 1.0 microM for alpha- and gamma-thrombin, respectively, at pH 7.0 [25 mM tris(hydroxymethyl)aminomethane, 0.15 M NaC1]. Fluorescence changes upon complexation included a considerable (approximately 30-nm) blue shift in the fluorescence emission maximum as well as a dramatic increase in the fluorescence emission intensity: a 70-fold enhancement was observed with alpha-thrombin vs. a approximately 220-fold enhancement with gamma-thrombin. Proflavin was not displaced upon bis-ANS binding. The unknown thrombin effectors ATP, Ca(II)ATP, Co(III)ATP, phosphate, and pyrophosphate bound with enhancement of the fluorescence of the bis-ANS-alpha-thrombin complex. The two inhibitors benzamidine and p-chlorobenzylamine as well as heparin caused decreases in bis-ANS-thrombin fluorescence: valerylamidine had no effect on the fluorescence of the bis-ANS-thrombin complex. Kinetic measurements with two chromogenic substrates, S-2238 and S-2160, indicated that bis-ANS acts as a partial noncompetitive inhibitor of thrombin amidase activity. The kinetic evidence combined with the ligand binding results suggests that bis-ANS does not overlap the catalytic site. The fluorophore ANS complexed with equal affinity to both alpha- and gamma-thrombins (Kd = 24 +/- 4 microM); however, the gamma-thrombin-ANS complex emission at 470 nm was enhanced 26% more than that for the alpha form.
D000255:Adenosine Triphosphate; D000345:Affinity Labels; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008024:Ligands; D013053:Spectrophotometry; D013917:Thrombin / Q000378:metabolism*
D000345:Affinity Labels; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008024:Ligands; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000255:Adenosine Triphosphate; D013917:Thrombin