Table 2. Temperature dependent association rate constants (kon) and thermodynamic values of the dye-labeled B7 binding to AV and SAV.
| Complexa | 25°C (×10-6M-1s-1) |
20°C (×10-6M-1s-1) |
15°C (×10-6M-1s-1) |
10°C (×10-6M-1s-1) |
EaForward (kcal/mol) | ΔSǂ (cal/°K·mol) | ΔGǂ (kcal/mol) |
|---|---|---|---|---|---|---|---|
| AV-BFl | 18.5 ± 2.0 | 10.7 ± 1.6 | 7.6 ± 2.0 | 4.86 ± 0.6 | 14.6 ± 0.6 | 34.9 ± 2.0 | 4.2 ± 0.5 |
| AV-BcO (pH 8)b | 9.5 ± 0.4 | 5.9 ± 0.4 | 3.9 ± 0.3 | 2.6 ± 0.3 | 14.4 ± 0.6 | 33.0 ± 2.5 | 4.6 ± 0.3 |
| AV-BcO (pH 9) | 7.9 ± 0.3 c | 5.4 ± 0.2 | 3.4 ± 0.1 | 2.3 ± 0.1 | 13.9 ± 0.7 | 30.9 ± 2.5 | 4.7 ± 0.1 |
| AV-BcO (pH 10) | 7.2 ± 0.3 c | 4.8 ± 0.2 | 3.0 ± 0.1 | 1.9 ± 0.1 | 14.9 ± 0.7 | 34.1 ± 1.3 | 4.7 ± 0.3 |
| 1) AV-B7 -DNAds*Fl | 15.7 ± 1.0 (30.5%) d |
11.2 ± 0.8 (68.9%) |
7.4 ± 0.7 (87.5%) |
4.2 ± 0.5 (95.0%) |
14.6 ± 0.8 | 34.8 ± 3.0 | 4.2 ± 0.2 |
| 2) AV- B7 -DNAds*Fl | 1.4 ± 0.1 (69.5%) |
0.98 ± 0.05 (31.1%) |
0.70 ± 0.03 (12.5%) |
0.52 ± 0.01 (0.05%) |
14.2 ± 0.5 | 29.0 ± 0.5 | 5.6 ± 0.6 |
| Average | NA | NA | NA | NA | 14.4 ± 0.2 | 32.8 ± 1.2 | 4.7 ± 0.3 |
| SAV-BFl | 74.7 ± 2.0 | 58.6 ± 1.6 | 53.1 ± 2.0 | 45.4 ± 1.0 | 5.3 ± 0.3 | 6.6 ± 0.5 | 3.3 ± 0.6 |
| SAV-BcO | 30.3 ± 2.0 | 24.0 ± 1.6 | 20.0 ± 1.2 | 17.3 ± 0.6 | 6.2 ± 0.4 | 7.8 ± 0.4 | 3.9 ± 0.4 |
| 1) SAV- B7 -DNAds*Fl | 53.0 ± 1.0 (44.4%) |
45.9 ± 0.8 (50%) |
36.4 ± 0.7 (51%) |
31.0 ± 0.5 (49%) |
6.2 ± 0.4 | 8.8 ± 0.6 | 3.5 ± 0.5 |
| 2) SAV- B7 -DNAds*Fl | 2.5 ± 0.1 (55.6%) |
2.3 ± 0.1 (50%) |
1.7 ± 0.1 (49%) |
1.50 ± 0.05 (51%) |
6.2 ± 0.5 | 3.1 ± 0.3 | 5.3 ± 0.6 |
| Average | NA | NA | NA | NA | 6.0 ± 0.2 | 6.6 ± 1.6 | 4.0 ± 0.6 |
The forward thermodynamic values (EaForward, ΔSǂ, Forward and ΔGǂ, Forward) were acquired from global fitting of the rate constants [42, 45] for the most probable model which resulted in a simple reaction with a transition state without intermediates. In the case of the B7-DNA duplex, the reaction model was a two-serial reaction model also with one transition state without intermediates. The nature of the serial reaction is probably caused by two B7 populations with different spatial orientations.
aThe probes were BFl, BcO, and B7 attached to a nucleotide in a 14-mer DNA duplex and the respective complex with AV and SAV.
bThe kon values were averaged from data in Table 1
cCalculated from an Arrhenius plot.
dThe pre-exponentials (in parenthesis) of kon1 and kon2 were renormalized after removing a third process associated with remaining photobleaching.