Table 3.
Metal Ion Content of Metallosubstituted HDAC8
| Molar ratio of metal:proteina,b | ||||||
|---|---|---|---|---|---|---|
| Fe | Co | Ni | Cu | Zn | Mn | |
| Native HDAC8c | 0.06 | —d | — | 0.13 | 0.87 | NDe |
| Co2+-HDAC8 | 0.02 | 1.1 | — | 0.01 | 0.02 | ND |
| Fe2+-HDAC8 | 0.79 | 0.08 | — | 0.08 | 0.05 | ND |
| Fe2+-D101L HDAC8 | 1.4 | — | — | 0.07 | 0.05 | ND |
| Fe3+-HDAC8 | 0.12 | — | — | 0.02 | 0.05 | — |
| Fe3+-D101L HDAC8 | — | — | — | 0.06 | 0.08 | — |
| Mn2+-HDAC8 | 0.02 | — | 0.02 | 0.06 | 0.25 | 1.1 |
| Mn2+-D101L HDAC8 | — | 0.05 | — | 0.04 | 0.06 | 0.98 |
| Cu2+-HDAC8 | 0.01 | — | — | 1.09 | 0.14 | ND |
| Ni2+-HDAC8 | ND | 0.02 | 0.14 | 0.08 | 0.13 | — |
a
Metallosubstituted HDAC8 was generated as described in the Materials and Methods. Metal-free enzyme was generated by dialyzing with EDTA. Each metal ion was subsequently introduced at a concentration of 100 μM chloride salt and dialyzed to a final concentration of 100 nM. Metal ion content of final protein samples was determined by ICP mass spectrometry.
b
The ratios of metal ion to protein was determined by dividing the metal ion concentration, as determined by ICP mass spectrometry, by the protein concentration, as determined by Bradford assay.
c
Native HDAC8 refers to recombinant protein expressed in E. coli with 100 μM ZnCl2.
d
“—” signifies a value of less than 0.01.
e
ND signifies not determined.