TABLE 2.
Mapping the epitope for mAb-112 by alanine scanning mutagenesis
Association (kon) and dissociation (koff) rate constants and the equilibrium dissociation constant (KD) were determined by global fitting of the SPR data to a 1:1 binding model. The following pro-uPA variants demonstrated binding kinetics indistinguishable from wild type: T9A, K15A, I16A, I17A, E20A, F21A, Y34A, R35A, R36A, H37A, R37aA, S37dA, T39A, Y40A, V41A, P60cA, K61A, K62A, E62aA, D63A, I65A, Y67A, R70A, R72A, L73A, N75A, N76A, Q78A, K82A, E84A, K92A, K110aA, E110bA, R110dA, K143A, N145A, S146A, T147A, D148A, L150A, P152A, E153A, Q154A,, D185A, Q185bA, W186A, K187A, D189A, S190A, Q192A, R217A, K223A, K225A, H241A, K243A, E244A.
| Pro-uPA variant | kon | koff | KD | KD-Variant/KD-wt | n |
|---|---|---|---|---|---|
| m−1s−1 | s−1 | nm | |||
| Wild type | (7.1 ± 2.8) × 104 | (6.1 ± 4.0) × 10−5 | 0.80 ± 0.29 | 1.0 | 5 |
| E144A | (1.3 ± 0.9) × 105 | (3.9 ± 1.8) × 10−4a | 3.1 ± 0.7a | 3.9 | 2 |
| Y149A | (1.2 ± 0.7) × 105 | (9.1 ± 0.3) × 10−3a | 93 ± 50a | 116 | 3 |
| Y151A | (1.9 ± 0.3) × 105 | (2.4 ± 0.6) × 10−2a | 124 ± 12a | 155 | 3 |
| K156A | (2.5 ± 0.5) × 105a | (1.3 ± 0.3) × 10−2 | 56 ± 19a | 70 | 4 |
| D194A | (6.4 ± 0.3) × 104 | (1.4 ± 0.1) × 10−3 | 22 ± 8a | 28 | 3 |
| F141A-G142A-K143A | (1.2 ± 0.2) × 105 | (7.1 ± 0.9) × 10−3 | 60 ± 5a | 75 | 2 |
Significantly different from corresponding value for wild type (p < 0.01).