TABLE 4.
Binding affinity of Fab C2 to the epitope I variants used in this studya
| Peptide | Sequenceb | KD (M) | Ka (1/s) | Kd (1/M · s) |
|---|---|---|---|---|
| C-epitope I | KKCQLINTNGSWHIC | 3.27 × 10−8 | 4.77 × 105 | 1.56 × 10−2 |
| L-epitope I | KKC(methyl)QLINTNGSWHIC(methyl) | NB | 0 | 0 |
| C-epitope I mutant I | KKCAAANTNGSWHIC | 4.53 × 10−7 | 1.22 × 106 | 5.53 × 10−1 |
| C-epitope I mutant II | KKCQLIAAAGSWHIC | 1.48 × 10−8 | 3.2 × 103 | 4.73 × 10−5 |
| C-epitope I mutant III | KKCQLINTNEEWHIC | 4.67 × 10−6 | 1.62 × 104 | 7.54 × 10−2 |
| C-epitope I mutant IV | KKCQLINTNGSAAAC | NB | 0 | 0 |
Association and dissociation rates and KD values were determined by SPR for the binding of peptides to Fab C2. Data were derived using BIAevaluation (version 4.2) software. Ka, association constant; Kd, dissociation constant; NB, no binding.
In C-epitope I and related mutants I to IV, a disulfide bridge connects the two cysteines. In L-epitope I, cysteines are methylated. Methylation is the minimum molecular modification required to block reactive thiols. C-epitope I mutants I, II, and IV were designed and prepared so that native residues were replace with alanines. In mutant III, glycine and serine were mutated to glutamic acid because alanines are too similar to glycine and serine.