Table 2.
Equilibrium dissociation constant (KD(eq)) values and association (kon) and dissociation (koff) rate constants determined for wildtype and N34S variant SPINK1 inhibitors against nonsulfated (purified from Escherichia coli) and native sulfated (purified from pancreatic juice) human trypsins
| SPINK1 | Hu1 (E. coli) | Hu1-SO4 (native) | Hu2 (E. coli) | Hu2-SO4 (native) |
|---|---|---|---|---|
| Wildtype | ||||
| kon (×106 M−1s−1) | 3.0 ± 0.3 | 3.5 ± 0.2 | 3.8 ± 0.2 | 2.4 ± 0.1 |
| koff (×10−6 s−1) | 8.2 ± 0.3 | 54.8 ± 6.2 | 0.5 ± 0.04 | 18.1 ± 0.8 |
| KD(calc) (pM) | 2.7 | 15.8 | 0.1 | 7.5 |
| KD(eq) (pM) | 1.1 ± 0.2 | 62.2 ± 10.3 | 0.3 ± 0.02 | 36.7 ± 2.3 |
| N34S | ||||
| kon (×106 M−1s−1) | 5.3 ± 0.2 | 2.7 ± 0.2 | 5.1 ± 0.2 | 3.1 ± 0.3 |
| koff (×10-6 s−1) | 7.0 ± 0.6 | 45.0 ± 5.1 | 0.4 ± 0.01 | 13.6 ± 0.5 |
| KD(calc) (pM) | 1.3 | 16.4 | 0.1 | 4.4 |
| KD(eq) (pM) | 1.5 ± 0.2 | 32.3 ± 2.9 | 0.4 ± 0.1 | 16.7 ± 0.8 |
Hu1, nonsulfated human cationic trypsin; Hu2, nonsulfated human anionic trypsin; Hu1-SO4, native sulfated human cationic trypsin; Hu2-SO4, native sulfated human anionic trypsin; KD(calc), equilibrium dissociation constant calculated from the rate constants.
Measurements were carried out as described under Experimental procedures section. Data from at least three experiments were fitted globally. The error of the fit is indicated.