Table 3.
Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions. EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare). Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days. Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column. The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions. The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time. The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization. The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.
| Method | Sample | Monomer area | Decamer area | Decamer/Monomer |
|---|---|---|---|---|
| Gel filtration Superdex 200 chromatography | EncFtnsH-MM | 64.3 | 583.6 | 0.1 |
| EncFtnsH-MM+Fe2+ | 1938.4 | 426.4 | 4.5 | |
| Analytical Gel filtration Day1 | EncFtnsH-decamer fractions | 20.2 | 1.8 | 11.2 |
| EncFtnsH-monomer fractions | 2.9 | 21.9 | 0.1 | |
| Fe(NH4)2(SO4)2/EncFtnsH-monomer | 11.0 | 13.0 | 0.8 | |
| FeSO4-HCl/EncFtnsH-monomer | 11.3 | 11.4 | 1.0 | |
| Analytical Gel filtration Day2 | EncFtnsH-monomer fractions | 8.3 | 22.8 | 0.4 |
| CoCl2/EncFtnsH-monomer | 17.7 | 14.5 | 1.2 | |
| MnCl2/EncFtnsH-monomer | 3.1 | 30.5 | 0.1 | |
| ZnSO4/EncFtnsH-monomer | 20.4 | 9.0 | 2.3 | |
| FeCl3/EncFtnsH-monomer | 3.9 | 28.6 | 0.1 | |
| Analytical Gel filtration Day3 | EncFtnsH-monomer fractions | 6.3 | 23.4 | 0.3 |
| MgSO4/EncFtnsH-monomer | 5.8 | 30.2 | 0.2 | |
| Ca acetate/EncFtnsH-monomer | 5.6 | 25.2 | 0.2 |