TABLE 1.
Kinetics of sCD14 binding to human Lf
| Parameter | Value |
|---|---|
| kass (M−1 s−1)a | 360,000 ± 110,000 |
| rb | 0.963 |
| Kd (nM)c | 16 ± 7 |
| kdiss (s−1)d | 0.0058 ± 0.0017 |
| Kd (nM)e | 45 ± 30 |
Result given as mean ± SE. The SE is derived from the deviation of the data from a one-site model and was calculated by matrix inversion using the FastFit software provided with the instrument. Each set of values of kon (e.g., Fig. 1) produced a value for kass and an associated SE. Data are pooled values from four experiments on three planar biotin surfaces with different amounts of immobilized hLf as described under “Materials and Methods”. The likehood of a one-site or a two-site model describing the association phase of each binding reaction was determined by four parameters of goodness of fit, as described previously (15). Firstly, if a one-site model could not fit the data, a two-site model was favored. Secondly, a direct statistical comparison of goodness of fit of a one-site and a two-site model was made for each binding curve using the F statistic; a two-site model was favored at a probability below 0.005. Thirdly, the randomness of the distribution of the data about the model was examined; a two-site model was favored over a one-site model when the data were nonrandomly distributed about the latter. Fourthly, the model had to yield realistic values, e.g., a positive value of kon at all concentrations of sCD14. In all experiments, at the lowest concentrations of sCD14 (<40 nM) a two-site model did not fit the data, whereas at higher concentrations of sCD14 a one-site model provided at least as good a fit as a two-site model.
The correlation coefficient of the linear regression through the values of kon for the binding model.
Kd was calculated from the kdiss/kass ratio, and the SE is the combined SE of the two kinetic parameters.
The kdiss is the mean ± SE of 10 values, obtained at high concentrations (1 μM) of sCD14.
Kd was calculated from the extent of binding observed near equilibrium at five or more different concentrations of ligate in four independent experiments. The SE is the combined error of the experiments.