TABLE 2.

Binding of l-ANT variants on heparin column and dissociation constants measured by TNS fluorescence titration

Protein	Heparin column affinitya		Dissociation constantb
	[NaCl] in peaks	[NaCl]WT − [NaCl]variant	DP8		LMW
			Kd	Kd/Kd,wt	Kd	Kd/Kd,wt
	mm	mm	nm		μm
WT	820	0	34 ± 1	1	0.17 ± 0.01	1
Cleaved	820	0	28 ± 1	0.80	0.18 ± 0.03	1.05
K76A	786	34	66 ± 2	1.93	0.21 ± 0.01	1.22
R79A	768	52	48 ± 04	1.38	0.20 ± 0.01	1.15
K144A	782	38	49 ± 3	1.44	0.24 ± 0.01	1.38
K145A	791	29	53 ± 3	1.54	0.24 ± 0.01	1.36
K146A	777	43	56 ± 6	1.63	0.28 ± 0.01	1.63
K148A	718	102	87 ± 1	2.54	0.44 ± 0.02	2.54
K151A	716	104	96 ± 5	2.80	0.52 ± 0.01	2.99
R155A	728	92	92 ± 1	2.67	0.31 ± 0.02	1.81
K162A	774	46	44 ± 1	1.28	0.17 ± 0.01	0.98
R163A	788	32	57 ± 1	1.62	0.18 ± 0.01	1.01
R380A	800	20	72 ± 2	2.10	0.33 ± 0.01	1.88
K438A	785	35	57 ± 1	1.66	0.20 ± 0.02	1.16
R440A	784	36	61 ± 7	1.79	0.16 ± 0.01	0.94
K158A/K151A/R155A	615	205	105 ± 3	3.04	1.62 ± 0.08	8.89

^a l-ANT variants were loaded onto a 1-ml heparin column and eluted with a 0.3–1.5 m NaCl gradient. The NaCl concentrations ([NaCl]) of the elution peaks are shown on the left.

^b The dissociation constant K_d values of l-ANT variants were determined at 0.15 m NaCl for DP8 and at 0.3 m NaCl for LMW heparin in the presence of 5–10 μm TNS in 50 mm Tris-HCl, pH 7.4, 20% glycerol, 0.1% polyethylene glycol 8000.