TABLE 4.

SPR analysis of binding of wild-type Nb4 or variants to active uPA or catalytically inactive uPA S195A

Association rate constants (k_on), dissociation rate constants (k_off), and the equilibrium dissociation constants (K_D) were determined by fitting the SPR data to a 1:1 binding model. Data are represented as mean ± S.D. for three experiments.

	kon	kon (WT)/kon (mut)	koff	koff (mut)/koff (WT)	KD	KD (mut)/KD (WT)
	μm−1·s−1		s−1 × 10−5		nm
uPA
Nb4	1.0 ± 0.2		5.4 ± 0.2		0.054 ± 0.02
uPA S195A
Nb4	1.8 ± 0.5		0.9 ± 0.4a		0.006 ± 0.003†
Nb4 D99A	0.026 ± 0.005b	70	290 ± 10b	322	110 ± 22b	20000
Nb4 R111A	0.023 ± 0.002b	78	180 ± 10b	200	78 ± 4b	14200
Nb4 Y113A	0.009 ± 0.002b	200	2.0 ± 0.7	2	2.3 ± 0.3b	420

^a Data are significantly different from the value of wild-type Nb4 binding to uPA (p < 0.05 by Student's t test).

^b Data are significantly different from the value of wild-type Nb4 binding to uPA S195A (p < 0.001 by Student's t test).