TABLE 2.
Binding affinity of gp5 variants with combinations of trx and trx-K36E
DNA polymerase activities were determined under “Experimental Procedures.” Reaction mixtures were incubated with 10 nm primed M13 ssDNA with [gp5] in linear range of 4 nm and titrating [trx] in the range from 1 to 6000 nm. DNA polymerase activities were measured by the incorporation of[3H]TMP at 37 °C over 3 min. Binding affinity was determined by using titration curves as shown in Fig. 2A for each gp5/trx complex to generate Scatchard plots. The observed equilibrium constant (Kobs) for each complex was determined as the negative slope of the corresponding plot. Comparison of bindings affinities (Kobs) of reconstituted gp5 variants with trx and trx-K36E is shown.
| trx | trx-K36E | |
|---|---|---|
| nm | nm | |
| gp5 | 197.9 ± 15 | 181.5 ± 22 |
| gp5-loop A | 247.5 ± 52 | 234.4 ± 22 |
| gp5-loop B | 134.4 ± 17 | 157.0 ± 18 |
| gp5-loop AB | 154.1 ± 16 | 149.1 ± 12 |