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. 2021 Mar 10;478(5):1085–1116. doi: 10.1042/BCJ20200838

Table 1. Optimisation of conditions for determining metal–protein affinities at [M]tot = 10 µM1.

No logKex2 [P]tot3 (µM) [MP]4 (µM) [ML] or [ML2]4 (µM) [L]tot (1 : 1)4 (µM) [L]tot (1 : 2)4 (µM) [MP]/[P]tot [ML]/[L]tot or 2[ML2]/[L]tot
1 −2.0 10 5.0 5.0 505 — 0.5 0.01
2 −1.0 10 5.0 5.0 55 — 0.5 0.09
3 0.0 10 5.0 5.0 10 — 0.5 0.5
4 1.0 55 5.0 5.0 10 — 0.09 0.5
5 1.0 10 5.0 5.0 — 717 0.5 0.01
6 2.0 10 5.0 5.0 — 234 0.5 0.04
7 3.0 10 5.0 5.0 — 81 0.5 0.12
8 4.0 10 5.0 5.0 — 32 0.5 0.3
95 4.7 10 5.0 5.0 — 20 0.5 0.5
↓
16		 ↓
5.0		 ↓
5.0
↓
25		 ↓
0.3		 ↓
0.4
10 6.0 230 5.0 5.0 — 25 0.02 0.4
1

[M]tot is decided by the detection sensitivity. Selection of [M]tot = 10 µM here is based on an assumption that the response of the selected probe is sensitive enough for a reliable quantification of the metal–probe complex MP in the system, but the [M]tot term may be scaled up or down along with [P]tot and [L]tot according to equations 8a and 8b, respectively. For example, the condition of logKex=0 for the stoichiometric competition of case 3 involving a 1 : 1 ML complex remains unchanged with the reaction re-scaling, but logKex for the same stoichiometric reaction of case 9 involved a 1 : 2 ML2 complex changes with [M]tot according to logKex=−log(2[M]tot) and thus the value logKex = 4.7 applies to the case of [M]tot = 10 µM only;

2

Kex=KD(P)/KD(L) for equation 7a or KD(P)β2 for equation 7b;

3

[P]tot ≥ [M]tot;

4

Calculated via equation 8a or 8b with the condition that ([MP]/[M]tot=0.5);

5

The experiment of case 9 with a molar ratio ([L]tot/[M]tot∼2) may run the risk of forming some 1 : 1 ML complex and it is generally advisable to set ([L]tot/[M]tot)≥2.5 to ensure ML2 complex formation: thus, a new set of conditions for case 9 may be re-set as the arrows suggest.