Table 2.
Association rate constants for ligands A to F that are obtained by analysing their simulated association profiles such as in Figure 3a, Figure 4 and Figure 5a
| Analysis of curve | kobs versus [L] | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| R 2 | Linear regression analysis | Analysis by simplified Strickland equation | ||||||||
| Ligand | [L] range (nM) | Monophasic | Biphasic | R 2 | kon M−1⋅min−1 | koff min−1 | R 2 | k3 (input) min−1 | k4 (input) min−1 | k2/k1 (input) nM |
| C (R'L) | 500–30 000 | 1 | 0.79 | 1 | 0.017 (0.016) | −6.5 × 10–4 (1.6 × 10–5) | 4.0 × 10–6 (4.0 × 10–6) | |||
| E (R'L) | 500–30 000 | 1 | 0.80 | 1 | 0.065 (0.064) | −5.5 × 10–4 (6.4 × 10–5) | 4.1 × 10–6 (4.0 × 10–6) | |||
| F (R'L) | 500–30 000 | 1 | 0.80 | 1 | 0.251 (0.250) | −2.8 × 10–4 (2.5 × 10–4) | 4.3 × 10–6 (4.0 × 10–6) | |||
| G (RL + RL) | 5–1000 | 0.93–0.98 | 1 | |||||||
| fast | 1 | 0.99 × 106 | 0.264 | |||||||
| slow | 0.82 | 1 | 0.016 (0.016) | 2.1 × 10–4 (2.6 × 10–4) | 2.6 × 10–7 (2.6 × 10–4) | |||||
| H (RL + R'L) | 5–500 | 0.92–0.98 | 1 | |||||||
| fast | 1 | 0.92 × 106 | 0.310 | |||||||
| slow | 0.99 | 1 | 0.068 (0.064) | −6.4 × 10–4 (1.0 × 10–3) | 3.2 × 10–7 (2.5 × 10–7) | |||||
| D (RL + R'L) | 5–500 | 1 | 1 | 0.99 × 106 | 9.39 × 10–3 | |||||
| A (R'L) | 5–500 | 1 | 1 | 0.94 × 106 | 3.95 × 10–3 | |||||
| B (RL + R'L) | 5–500 | 1 | 1 | 1.00 × 106 | 3.19 × 10–3 | |||||
Ligands are presented by order of appearance in the text. Bound species that are taken in consideration for the analysis are also mentioned. In this respect, it is assumed that RL dissociates so swiftly for ligands A, C, E and F that even at the earliest obtainable time points, it is no longer detectable when performing classical filtration radioligand binding assays. Data points are collected (≥10 per curve) and first analysed according to a monophasic exponential association model (Prism 4.0 by GraphPad Software Inc., San Diego, CA, USA). R 2 is represented as 1 in the table when above 0.999; if less curves are further analysed according to a biphasic exponential association model (in Prism 4.0 by GraphPad Software Inc.). All kobs versus [L] relationships are then checked for their linearity. When the plots are linear (i.e. R 2 = 1), they yield kon from the slope of the kobs versus [L] plot and koff from the extrapolated kobs for [L] = 0. When R 2 < 1, the plots are analysed according to the simplified Strickland equation (Prism 4.0 by GraphPad Software Inc. with equation 5 in Figure 1b as input) to yield k3, k4 and the k2/k1 ratio (Strickland et al., 1975). For comparison, the values of the microkinetic rate constants (or their ratio) that were utilized as input for the simulations are also given in brackets.