Table 2. Dissociation constants of ORF56 as measured by fluorescence anisotropy titrations.

DNA	Buffer	Kd (nM)	Kc (nM)a	Specificityb
wt-F	Tris–HCl	1.5 (0.63–2.45)c
	Na cacodylate	0.4d
wt	Tris–HCl	0.88 ± 0.11e
rep	Tris–HCl	210 ± 13f
CT DNA	Tris–HCl		670 ± 70	270
CT DNA (53°C)	Tris–HCl		1220 ± 100	1500

The buffer in all experiments was 20 mM Tris–HCl, pH 7.5, 100 mM KCl, 0.01% Tween 20. 20 mM Tris–HCl was substituted for 20 mM sodium cacodylate where indicated. wt-F is the fluorescently labelled wt DNA. Rep is a single repeat DNA, CT DNA is sonicated calf thymus DNA. K_d is the dissociation constant of the equilibrium (ORF56)₄ + DNA ⇔ DNA·(ORF56)₄. The K_d value was obtained by least square fitting of the binding isotherm and refers to the tetramer concentration.

^aDetermined from a competition experiment. The dissociation constant K_c refers to the microscopic dissociation constant for all non-specific sites on calf thymus DNA.

^bSpecificity was calculated by K_c/K_d. K_d at 25°C was 2.45 ± 0.23 nM and at 53°C 0.8 ± 0.2 nM.

^cRange of six independent experiments.

^dAverage of two experiments.

^eDetermined from a competition assay.

^fDetermined from a competition assay. The dissociation constant applies to a dimer half-site equilibrium.