TABLE 1.

Summary of apparent kinetic constants for protein binding to model DNA P/T^a

Protein	KCl concn (mM)	Nitrocellulose filter-binding assayb				Biosensor assayc		Enzyme kinetics assaydKd (nM)
		kon (M−1 s−1) (koff/Kd)	koff (s−1)	t1/2 (min)	Kd (nM)	Kd (nM) [koff/kon]	Rel. Kd
UL42	50	NDe	ND	ND	ND	35.3	2.1	ND
	125	ND	ND	ND	ND	150,000	8,770	ND
Pol	50	1.87 × 104	1.55 × 10−3	7.5	83.0	17.1	1.0	44.2
	125	If	I	I (very fast)	I	47.7	2.8	ND
Pol/UL42	50	1.27 × 105	8.37 × 10−4	13.8	6.60	9.02	0.5	ND
	125	1.16 × 105	8.76 × 10−4	13.2	7.51	30.7	1.8	7.43

^aAll kinetic constants are apparent except for those determined by pre-steady-state kinetic analysis.

^bValues for K_d and k_off were determined as described in the legends to Fig. 4 and 5, respectively. The values of t_1/2 were derived from the equation ln 0.5 = −kt_1/2, and the value of k_on was determined according to equation 2.

^cAll kinetic constants were derived by global fitting of the binding and dissociation curves from five different protein concentrations using the Biaevaluation version 3.0 software. All binding assumed the langmuir model A + B = AB. Rel. K_d, relative or normalized K_d.

^dDerived by active-site titration of enzyme activity on the model P/T under pre-steady-state conditions (Chaudhuri and Parris, unpublished).

^eND, not done.

^fI, indeterminate.