Table 1.
Binding affinities between the CXCR4 ectodomain peptides and MIF versus CXCL12 as determined by fluorescence titration spectroscopy, fluorescence polarization (FP), and microscale thermophoresis (MST).
| CXCR4 ectodomain (ECD) peptide | Fluos (TAMRA)-ECD peptide/MIFa, b (app. KD [nM][c]) | Alexa-MIF/ECD peptided (app. KD [nM]) | Fluos (TAMRA)-ECD/CXCL12e (app. KD [nM]) |
|---|---|---|---|
| Fluorescence spectroscopy | |||
| msR4M-L1 | 40.7 ± 4.0 | 31.1 ± 16.6 | >6340 |
| msR4M-L2 | 18.6 ± 2.9 | 40.5 ± 7.6 | >6340 |
| msR4M-L1ox | 28.9 ± 2.5 | 30.0 ± 6.3 | 84.6 ± 42.1 |
| msR4M-L2ox | 105.3 ± 44.9 | 59.6 ± 15.3 | 54.8 ± 10.3 |
| msR4M-LS | 6.9 ± 2.0 | n.d. | 17.4 ± 4.7 |
| ECL1 | n.d. | 345.2 ± 79.4 | n.d. |
| ECL2 | >5000 | 2458 ± 1054 | n.d. |
| Fluorescence polarization (FP) | |||
| msR4M-L1 | 24.4 ± 5.3 | 10.6 ± 1.2 | >2500 |
| Microscale thermophoresis (MST) | |||
| msR4M-L1 | 77.2 ± 37.1 | n.d. | >3125 |
aFor fluorescence spectroscopy and FP, Fluos-labeled ECD peptides were used at a concentration of 5 nM
b For MST, TAMRA-labeled ECD peptide was used at a concentration of 100 nM
c Reported apparent KD values are means ± SD from 3 (fluorescence spectroscopy and FP) or 5 (MST) independent experiments and were calculated as described in Methods
d Alexa-MIF was used at a concentration of 10 nM
eAlexa-CXCL12 measurements were not pursued, because of the notion that Alexa labeling could interfere with the crucial residue Lys-1 of CXCL1230 as well as other binding-relevant lysines. ECD, extracellular domain; app., apparent; n.d., not determined