TABLE 1.

Binding kinetics of Hh ligands to ch5E1 Fab in the presence and absence of divalent ions by biolayer interferometry

Binding measurements for human and rat Shh were carried out using immobilized Hh ligand and ch5E1 or m5E1 Fabs in solution. Binding measurements for human Ihh and human Dhh were carried out using immobilized ch5E1 and Hh ligand in solution as described under “Experimental Procedures.”

Hh ligand	Divalent metal cation	KD	kon	koff
		nm	m−1s−1	s−1
Shh	None	6.94 (6.16)a	5.07 × 105	35.2 × 10−4
Shh	Zn2+	4.82 (5.23)	5.71 × 105	27.5 × 10−4
Shh	Ca2+	1.12 (1.46)	2.58 × 105	2.89 × 10−4
Rat Shhb	Zn2+, Ca2+	1.51	3.67 × 105	5.55 × 10−4
Shh	Zn2+, Ca2+	0.31 (0.67)	2.09 × 105	0.65 × 10−4
Ihh	Zn2+, Ca2+	0.29	3.55 × 105	1.05 × 10−4
Dhh	Zn2+, Ca2+	1.71	4.95 × 105	8.47 × 10−4

^a Values in parentheses are for the parental murine 5E1 Fab.

^b Rat Shh refers to the sequence as described in Roelink et al. (43).