Table 3.
SEC-MALS derived molecular masses for TcdA-A2 + VHH complexes.
| Sample | Tr (min) | Mr (kDa) | Mobs (kDa) | Species | Relative proportion (%)a,b |
|---|---|---|---|---|---|
| TcdA-A2 | 3.748 ± 0.001 | 32.1 | 31.3 ± 1.1 | Ag | 100 |
| A20 | 3.864 ± 0.001 | 16.3 | 17.9 ± 0.1 | Ab | 100 |
| A26 | 3.866 ± 0 | 16.0 | 18.5 ± 2.1 | Ab | 100 |
| A20-A20 | 3.786 ± 0.001 | 28.7 | 28.6 ± 2.6 | Ab | 100 |
| A26-A26 | 3.665 ± 0.001 | 29.4 | 25.4 ± 0.6 | Ab | 100 |
| A20-A26 | 3.786 ± 0.001 | 29.0 | 29.6 ± 0.6 | Ab | 100 |
| A26-A20 | 3.745 ± 0.001 | 29.0 | 29.2 ± 0.2 | Ab | 100 |
| A20-A26 (G4S) | 3.792 ± 0.001 | 28.5 | 26.1 ± 0.5 | Ab | 100 |
| A20-A26 (G4S)2 | 3.767 ± 0.002 | 28.8 | 24.1 ± 1.0 | Ab | 100 |
| TcdA-A2 + A20 | 3.493 ± 0.001 | 48.4 [Ag.Ab] | 41.1 ± 0 | Ag.Ab | 79.6 |
| 3.974 ± 0.001 | 16.3 [Ab] | 19.5 ± 0.5 | Ab | 16.3 | |
| – | – | – | – | 4.1 | |
| TcdA-A2 + A26 | 3.470 ± 0 | 48.1 [Ag.Ab] | 41.1 ± 0.2 | Ag.Ab | 69.2 |
| 3.969 ± 0 | 16.0 [Ab] | 16.4 ± 0.7 | Ab | 27.0 | |
| – | – | – | – | 3.7 | |
| TcdA-A2 + A20-A20 | 3.145 ± 0.004 | 92.9 [(Ag)2.Ab] | 77.3 ± 0.9 | (Ag)2.Ab | 92.4 |
| 3.754 ± 0.002 | 60.8 [Ag.Ab] | 65.1 ± 5.9 | Ag.Ab | 7.5 | |
| TcdA-A2 + A26-A26 | 3.060 ± 0.001 | 93.6 [(Ag)2.Ab] | 73.8 ± 0.6 | (Ag)2.Ab and/or Ag.Ab | 76.3 |
| 61.5 [Ag.Ab] | |||||
| 3.600 ± 0.003 | 61.5 [Ag.Ab] | 43.5 ± 1.0 | Ag.Ab and/or Ag | 23.7 | |
| 32.1 [Ag] | |||||
| TcdA-A2 + A20-A26 | 3.479 ± 0 | 61.1 [Ag.Ab] | 55.1 ± 0.2 | Ag.Ab | 91.2 |
| – | – | – | – | 4.7 | |
| – | – | – | – | 3.3 | |
| TcdA-A2 + A26-A20 | 3.088 ± 0.001 | 122.2 [(Ag)2.(Ab)2] | 108.4 ± 0.8 | (Ag)2.(Ab)2 | 54.6 |
| 3.269 ± 0 | 93.2 [(Ag)2.Ab] | 102.6 ± 2.0 | (Ag)2.Ab and/or Ag.(Ab)2 | 42.7 | |
| 90.1 [Ag.(Ab)2] | |||||
| – | – | – | – | 2.1 | |
| TcdA-A2 + A20-A26 (G4S) | 3.077 ± 0.002 | 121.4 [(Ag)2.(Ab)2] | 103.3 ± 0.6 | (Ag)2.(Ab)2 | 46.9 |
| 3.438 ± 0.002 | 60.6 [(Ag.Ab)] | 66.8 ± 2.0 | Ag.Ab | 53.0 | |
| TcdA-A2 + A20-A26 (G4S)2 | 3.065 ± 0.003 | 121.8 [(Ag)2.(Ab)2] | 116.6 ± 0.3 | (Ag)2.(Ab)2 | 9.2 |
| 3.436 ± 0.003 | 60.9 [(Ag.Ab)] | 56.4 ± 0.6 | Ag.Ab | 90.8 |
Ag, TcdA-A2; Ab, VHH monomer or dimer depending on sample; Tr, column retention time; Mr, theoretical molecular mass; Mobs, observed molecular mass determined by MALS UV. Values are mean ± SD with from two technical replicates. aTotal may not add to 100% in some cases, due to minor peaks not identified in the analysis. Relative proportion is only for the representative data plotted in Figure 4. bPeaks with <5% relative proportion were omitted from species assignment.