This method allows undergraduate students to rapidly purify iron-binding metalloenzymes and then use that protein to answer key questions about structure and function, all within the confines of the teaching laboratory. The main advantage of this technique is that it is applicable to non-heme iron-binding enzymes. It is accessible to undergraduate students, and it's readily implemented in the teaching laboratory.
To begin this procedure, obtain a 1.5-by-20 centimeter column equipped with a luer lock outlet, an upper cap that also contains a luer lock fitting, and a lower bed support to retain resin particles. Fit the lower luer lock fitting with a stopcock to control flow. Next, obtain a bottle containing a 50%slurry of nickel-bound nitriloacetic acid resin in 20%ethanol that was previously stored at four degrees Celsius.
Gently swirl the bottle to evenly re-suspend the resin. Working at room temperature, use a graduated pipette to withdraw two millimeters of the slurry and transfer it to the column. After the excess storage solution drains from the resin, use a Pasteur pipette to carefully add at least 30 milliliters of chilled lysis bind buffer, making sure not to disturb the resin surface.
Run the buffer down the walls of the column to prevent splashing. Equilibrate the resin by allowing the buffer to drain slowly from the column by gravity into a collection beaker. To begin, prepare the nickel-NTA column for the addition of the cell-free crude extracts by opening the stopcock and allowing the remaining lysis binding buffer to drain by gravity through the nickel-NTA resin.
When the buffer has drained completely and the resin surface is exposed, close the stopcock. Next, use a Pasteur pipette to carefully pipette the cell-free crude extracts onto the resin, taking care not to disturb the surface. Run the crude extracts down the wall of the column to avoid splashing.
After all the crude extracts have been transferred to the column, open the stopcock so that the column can drain by gravity. Collect 100 microliters of the flow-through for later analysis. Use a short length of plastic tubing to connect a 10 milliliter luer lock syringe to the cap of the column.
Draw out the syringe plunger and then tightly fill the column cap onto the top of the column. Gently compress the plunger while collecting the eluent into a graduated cylinder to manually assess the flow rate. Rates between one and two milliliters per minute are compatible with the resin and the experimental setup.
Gently increase compression of the syringe plunger as the flow rate slows. When the meniscus of the applied liquid reaches five to 10 milliliters above the resin bed, remove the column cap and attached hand pump to prevent the introduction of air into the resin. Let the remaining volume drain by gravity.
After the crude extracts have finished draining and the resin surface is exposed again, use a Pasteur pipette to carefully add about 30 milliliters of chilled wash buffer to the column, being careful not to disturb the resin surface. Open the stopcock and let the wash buffer drain through the column. While the column is draining, label 16 microcentrifuge tubes for fraction collecting.
When the buffer has completely drained and the resin surface is exposed again, close the stopcock. Using a Pasteur pipette, carefully add about 30 milliliters of chilled elution buffer to the column, taking care not to disturb the surface of the resin. Then, open the stopcock slowly to allow the elution buffer to elute the polyhistidine-tagged protein from the column.
Collect one milliliter of eluent in each of the marked microcentrifuge tubes. After testing for protein, combine the protein-containing fractions into a clean conical tube. Proceed with a reconstitution of the metal ion cofactor, or freeze the protein in three milliliter aliquots at minus 80 degrees Celsius.
To begin the reconstitution of the metal, put a three milliliter aliquot of the purified enzyme, suspended in elution buffer, on ice. Using the volume of protein in the tube, calculate the amount of sodium ascorbate and dithiothreitol necessary such that the final concentration of each is 12.5 millimolar in the sample. Add the solid sodium ascorbate and dithiothreitol, and gently but thoroughly mix to dissolve completely.
Tap a few one milliliter granules of solid iron sulfate heptahydrate out onto a piece of weigh paper. Fold the paper over the granule and crush it with the flat side of a metal spatula. Add a grain of the resulting powder to the tube containing the protein solution.
Vortex to mix, resulting in a rosy-pink color. Cap the tube, and incubate the pink solution on ice for between 10 and 30 minutes. The pink color will fade over time.
For gel filtration, use a gel filtration column packed with 10 milliliters of spherical polyacrylamide gel in a 1.5-by-12 centimeter column, which is also equipped with a 10 milliliter reservoir. Then equilibrate the column as outlined in the text protocol. When the buffer has drained from the column and the upper bed support is exposed, close the stopcock.
Pipette the three milliliter solution of iron two reconstituted metalloenzyme onto the exposed upper bed support. Open the stopcock, and let the entire three milliliter sample enter the column by gravity. Discard the first three milliliters of eluent.
Any pink color that forms while handling the solution will be trapped at the top of the column. When the column has stopped dripping and the upper bed support is exposed, add four milliliters of the gel filtration buffer, and collect four milliliters of eluent over 8.5 milliliter fractions. After combining protein-containing fractions, proceed with the assay or storage of the sample as outlined in the text protocol.
In this study, students purified and subsequently reconstituted a polyhistidine-tagged non-heme iron-binding dioxygenase. SDS-PAGE analysis shows that the tagged protein is effectively purified. Student-collected activity data on the enzymatic action of purified L-dopa dioxygenase after reconstitution with iron two and subsequent gel filtration is shown here.
The five second lag before data collection begins is typical of students executing this technique for the first time. The steady-state assay indicates that the kinetic parameters, as determined by fitting the progress curves, are consistent with published results. While attempting this procedure, it's important to remember that more iron is not better, as too much iron will cause protein precipitation.
Iron concentrations in three-to-five molar excess of the protein are sufficient. Following this procedure, students can perform additional experiments to measure solution structure, look at enzyme or substrate-dependent chemistry, all to answer additional questions about reaction mechanism.
