Table 5.
Iron loading capacity of EncFtn, encapsulin and ferritin. Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air. Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively. Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein. Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold). The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin. Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays. The proteins used in Fe loading experiment came from a single preparation.
| Protein sample | Fe(NH4)2(SO4)2 loading (µM) | Fe detected by ferrozine assay (µM) | Protein detected by BCA microplate assay (µM) | Fe / monomeric protein | Maximum Fe loading per biological assembly unit |
|---|---|---|---|---|---|
| 8.46 µM EncFtnsH-10mer | 0 | 4.73 ± 2.32 | 5.26 ± 0.64 | 0.90 ± 0.44 | |
| 39.9 | 9.93 ± 1.20 | 5.36 ± 0.69 | 1.85 ± 0.22 | ||
| 84 | 17.99 ± 2.01 | 4.96 ± 0.04 | 3.63 ± 0.41 | ||
| 147 | 21.09 ± 1.94 | 4.44 ± 0.21 | 4.75 ± 0.44 | 48 ± 4 | |
| 224 | 28.68 ± 0.30 | 3.73 ± 0.53 | 7.68 ± 0.08 | ||
| 301 | 11.27 ± 1.10 | 2.50 ± 0.05 | 4.51 ± 0.44 | ||
| 8.50 µM Encapsulin | 0 | -1.02 ± 0.54 | 8.63 ± 0.17 | -0.12 ± 0.06 | |
| 224 | 62.24 ± 2.49 | 10.01 ± 0.58 | 6.22 ± 0.35 | ||
| 301 | 67.94 ± 3.15 | 8.69 ± 0.42 | 7.81 ± 0.36 | ||
| 450 | 107.96 ± 8.88 | 8.50 ± 0.69 | 12.71 ± 1.05 | ||
| 700 | 97.51 ± 3.19 | 7.26 ± 0.20 | 13.44 ± 0.44 | ||
| 1000 | 308.63 ± 2.06 | 8.42 ± 0.34 | 36.66 ± 0.24 | 2199 ± 15 | |
| 1500 | 57.09 ± 0.90 | 1.44 ± 0.21 | 39.77 ± 0.62 | ||
| 2000 | 9.2 ± 1.16 | 0.21 ± 0.14 | 44.73 ± 5.63 | ||
| 8.70 µM EncFtn-Enc | 0 | 3.31 ± 1.57 | 6.85 ± 0.07 | 0.48 ± 0.23 | |
| 224 | 116.27 ± 3.74 | 7.63 ± 0.12 | 15.25 ± 0.49 | ||
| 301 | 132.86 ± 4.03 | 6.66 ± 0.31 | 19.96 ± 0.61 | ||
| 450 | 220.57 ± 27.33 | 6.12 ± 1.07 | 36.06 ± 4.47 | ||
| 700 | 344.03 ± 40.38 | 6.94 ± 0.17 | 49.58 ± 5.82 | ||
| 1000 | 496.00 ± 38.48 | 7.19 ± 0.08 | 68.94 ± 5.35 | 4137 ± 321 | |
| 1500 | 569.98 ± 73.63 | 5.73 ± 0.03 | 99.44 ± 12.84 | ||
| 2000 | 584.30 ± 28.33 | 4.88 ± 0.22 | 119.62 ± 5.80 | ||
| 8.50 µM Apoferritin | 0 | 3.95 ± 2.26 | 9.37 ± 0.24 | 0.42 ± 0.25 | |
| 42.5 | 10.27 ± 1.12 | 8.27 ± 0.30 | 1.24 ± 0.18 | ||
| 212.5 | 44.48 ± 2.76 | 7.85 ± 0.77 | 5.67 ± 0.83 | ||
| 637.5 | 160.93 ± 4.27 | 6.76 ± 0.81 | 23.79 ± 3.12 | 571 ± 75 | |
| 1275 | 114.92 ± 3.17 | 3.84 ± 0.30 | 29.91 ± 2.95 | ||
| 1700 | 91.40 ± 3.37 | 3.14 ± 0.35 | 29.13 ± 3.86 |