The Michaelis-Menten (Km ), catalytic (kcat ), and specificity (kcat/Km ) constants were determined for humSft α- and β-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA), Chromozym-TH (Tos-Gly-Pro-Arg-pNA), and Spectrozyme-TH (H-D-HHT-Ala-Arg-pNA) in 0.15 M NaCl buffered with 10 mM HEPES ancTlO mM Tris-HCT at pH 7.4, 37°C. Spontaneous hydrolysis was insignificant for these substrates. Both S-2238 and Spectrozyme-TH exhibited limiting solubilities at ∼35 μM, while Chromozym-TH did not do so up to 50 μM. From initial estimates, Km's and k t's were refined by computer Gause-Newton iterations or nonlinearneast-square fits for the concentration of p-nitroanalide formed per second versus the initial substrate concentration. No major differences were found between a-thrombin (99% α, 91% esterolytically active enzyme, and > 3,500 kilo-U.S. clotting units/g with fibrinogen) and Yγ-thrombin (98% γ. 89% active enzyme, and < 10 kilo-units/g). For α- versus γ-thrombin and the three substrates, respectively, the Km 's were 6.75 ± 0.13 vs 7.62 ± 0.30, 18.4 ± 0.4 vs 23.0 ± 0.3, and 2.53 ± 0.02 vs 3.85 ± 0.51 μM; the kcat> s were 125 ± 1 vs 134 ± 2, 181 ± 2 vs 130 ± 1, and 35.5 ± 0.2 vs 52.4 ± 2.0 s−1 ; and the kcat/Km 's were 18.5 vs 17.6, 9.84 vs 5.65, and 10.1 vs 13.6 s−1 μm . These values closely approximate those of 7.2 ± 0.9 μM, 84 ± 4 s−1 , and 11.7 s−1 μM−1 determined by Higgins, Lewis, and Shafer (J. Biol. Chem. 258:9276-9282, 1983) for the Aα cleavage of human fibrinogen by human α-thrombin under physiologically relevant conditions. Thus, these chromogenic substrates have thrombin specificities similar to that of fibrinogen, although their amidolytic activities are independent of additional active-site regions required for fibrinogen clotting activity (α vs γ-thrombin). Fibrinogen interactions with such active-site regions might account for why the fibrinogen Aa site is an atypical thrombin susceptible bond and the high species variability of fibrinopeptides. (Supported in part by NIH grant HL-13160).
Using Enzyme-Linked Markers with Chromogenic Substrates in Expansion Microscopy
