Table 2. Kinetics and affinity of PCNA interaction with cytosolic components of NADPH-oxidase.

Immobilized ligand/soluble analyte	PCNA
	ka1 (M−1 s−1)		kd1 (s−1)	kforward (s−1)	kbackward (s−1)	KD (M)
p47phox (two-state model)	8.7 × 103		8.4 × 10−3	7.5 × 10−3	6.5 × 10−4	7.7 × 10−8
p47-PX domain (1:1 model)	85.2		2.8 × 10−3	NA	NA	3.3 × 10−5
p47 peptide 106–127	1.0 × 104		0.35	NA	NA	3.4 × 10−5 (KD1)
(Heterogeneous ligand model)	160		0.0111	NA	NA	6.9 × 10−5 (KD2)
p47 peptides
83–110	ND		ND	ND	ND	ND
63–90
46–67
Rac2 (two-state model)	541		17 × 10−3	5.7 × 10−3	6.6 × 10−4	3.3 × 10−6

No significant binding was detected for p40phox and p67phox for concentrations ≤1 µM. The binding of PCNA with p47phox (25–400 nM), p47-PX domain (1.83–29.5 µM), or p47peptide 106–127 (0.31–40 µM) was investigated by SPR as described in Materials and methods. Kinetics parameters were calculated using either two-state, 1:1 Langmuir, or heterogeneous ligand reactions models. For the two-state binding model (A + B ↔ AB ↔ AB*) that fit the best for full-length p47phox, k_a1, k_d1, k_forward, and k_backward constants were determined by global fitting. The dissociation constant K_D was determined from the (k_d1/k_a1)/(1 + k_forward/k_backward) ratio. The heterogeneous ligand model accounts for two different binding sites on the immobilized PCNA, and the corresponding two kinetic values were calculated. For 1:1 Langmuir and heterogeneous ligand models, the equilibrium dissociation constant is calculated by K_D = k_d/k_a. The data presented were obtained with a statistic χ² value <2. Rac2, p40phox, and p67phox were also tested for their interaction with PCNA. Binding of Rac2 (0.51–8, 2 µM) to PCNA was measured and evaluated as reported below (χ² value 0.6). NA, not applicable; ND, the very low binding signal detected for p47 83–110, 63–90, and 46–67 peptides did not allow evaluation of kinetic data.