Table 2.

Michaelis-Menten kinetics of Spectrozyme TH hydrolysis by α-thrombin in the presence of the trimer 9a.^a

[9a] (μM)	Overnight incubation at 25°C		2 hr incubation at 37°C
	KM (μM)	VMAX (mAU/min)	KM (μM)	VMAX (mAU/min)
0	9.5 ± 1.8b	81 ± 5	7.7 ± 2.0	86 ± 7
0.36	11.1 ± 2.5	80 ± 6	–c	–
0.63	7.7 ± 1.3	69 ± 3	6.7 ± 2.3	72 ± 8
1.53	–	–	2.2 ± 0.8	30 ± 2
1.80	3.2 ± 0.5	35 ± 1	–	–
3.60	3.1 ± 0.60	19 ± 1	2.7 ± 0.6	25 ± 1
9.90	–	–	1.9 ± 0.6	15 ± 0.8

^aK_M and V_MAX were measured by monitoring the initial rate of thrombin hydrolysis of Spectrozyme TH (2 – 80 μM) from the linear increase in absorbance at 405 nm in the presence of fixed concentration of 9a that was incubated either overnight at 25°C or for two hours at 37°C in 20 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM CaCl₂ and 0.1 % PEG8000. The data was fitted by the standard Michaelis – Menten equation to obtain K_M and V_MAX, as described in ‘Experimental Procedures’.

^bError represents ±1 S.E.

^cNot measured