Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.
This method is designed to answer key questions in the field inorganic biochemistry, about how to maintain enzyme activity in proteins that contain oxygen-sensitive metals. The main advantage of this technique is that it helps you visualize ways in which you can maintain a protein's metal in its reduced state. Generally, individuals new to this technique will struggle because it is difficult to maintain materials and reagents to be completely anaerobic.
24 hours after inducing protein expression, collect the cells by centrifugation, and resuspend the bacterial cell pellet in 20 milliliters of amylose column buffer per 1.5 liters of growth. Add one milligram of lysozyme to the cell suspension, and stir the cells for 20 minutes on ice. At the end of the incubation, lyse the resuspended cell solution via high-pressure homogenization with three to five passes at approximately 15, 000 psi, and transfer the protein to a 50-milliliter centrifuge tube.
Flush the headspace with nitrogen for one minute, and centrifuge the cell lysate to remove the insoluble debris. Transfer the supernatant to at least one 50-milliliter tube, and cap the lysate with a rubber septum. Using a 20-gauge needle, slowly bubble nitrogen gas through the solution for two minutes to displace the oxygen.
Then, wrap the septum with paraffin film, secure the Parafilm with copper wire, and place the supernatant into the glove box with the column materials. When the solvent is just above the resin bed, in a previously prepared amylose column, load 50 milliliters of protein supernatant onto the column, collecting the flow-through in five-milliliter fractions under moderately pressurized nitrogen at approximately five milliliters per minute. When all the flow-through has been eluted, wash the column with another three column volumes of amylose column buffer.
Add five CV of elution buffer, and then manually collect three-milliliter fractions in glass test tubes under moderately pressurized nitrogen. Test the solutions for oxygen presence with ferrous ammonium sulfate and dithiothreitol. The solution will turn dark blue or black if oxygen is present, like the tube on the right.
The presence of oxygen will result in purified protein with low catalytic activity. Solutions with minimal oxygen present will be lavender or light blue translucent color, like the tube on the left. Next, use an external nitrogen line to pressurize the stirred cell chamber, and concentrate the protein to 50 milliliters at a moderate stirring speed two times.
Then, concentrate the protein to 10 milliliters, and filter the concentrated protein through a 0.45-micrometer-pore syringe filter. Aliquot 150 microliters of protein into individual 200-microliter polymerase chain reaction tubes. Then, remove the capped aliquots from the glove box for immediate flash-freezing in liquid nitrogen and storage at minus 80 degrees Celsius.
For DesB desalting, first add three milliliters of Sephadex G-25 Coarse desalting gel to a nine-milliliter borosilicate column, and wash the column with two column volumes of degassed desalt buffer. When the DesB has thawed, load the purified protein onto the column via a gravity-controlled process, and discard the flow-through before eluting three drops of protein into each of 12 vials, with approximately five milliliters of desalting buffer. To prepare the oxygen electrode, use scissors to cut one approximately 1.5-inch-square of spacer paper, and cut small triangles onto each face of the square, with slits in the corners to aid in the wrapping of the electrode.
Add five drops of a 50%potassium chloride solution onto the silver anode groove of the electrode, and connect the drops so they form a continuous ring of solution. Add two drops of the potassium chloride solution to the top of the platinum electrode, and carefully place the spacer paper on top of the platinum electrode, smoothing out the spacer paper so there are no air bubbles. Cut an approximately two-inch-long piece of S4 30m PTFE membrane, and place it on top of the spacer paper, removing the edges of the membrane so they are aligned with the outer ring of the electrode.
Using an O-ring applicator, push a small O-ring over the electrode to secure the spacer paper and membrane onto the electrode, and place a larger O-ring onto the circular groove of the electrode, taking care that there is no membrane underneath. Slide the top chamber of the electrode onto the base, and screw the two pieces together. Place the assembled electrode on its base, and connect the electrode to the monitoring system.
To determine the rate of enzymatic reaction, use an oxygen-sensitive Clark-type electrode, with computer integration to measure the oxygen consumption via the electrode control unit. Add one milliliter of Tris buffer in the appropriate volume of substrate to the electrode chamber, and stir the solution for 10 seconds. After 10 seconds, slowly seal the chamber with the plunger, and screw the top down.
Use a clean tissue to soak up the excess liquid that is displaced from the chamber, and allow the electrode to equilibrate where it can produce a stable measurement of the oxygen concentration in the solution for at least one minute. Next, use a gas-tight, 10-microliter syringe to add approximately one microliter of enzyme to the electrode chamber through the plunger, and collect the rate data. When all of the rate for a given substrate concentration has been collected, use a plastic tube connected to a side-arm vacuum flask to empty the electrode chamber, and repeatedly rinse the chamber for four to six cycles with water.
Then, set up the solution in the electrode as just demonstrated, using a new substrate concentration. SDS-PAGE gel analysis of individual fractions from DesB-maltose binding protein fusion construct purification reveals the isolation of a pure protein product, aside for the presence of DesB and maltose binding protein domain products that cleaved from each other. Once all the kinetic measurements have been taken, the activity of DesB with gallate, for example, can be obtained by measuring the rate of oxygen consumption in varying gallate concentrations.
Conversely, the rate of inhibition of DesB with four-nitrocatechol can be determined by observing the reduction in the oxygen consumption rate of DesB with one-millimolar gallate in the presence of varying concentrations of four-nitrocatechol, indicating, for example, that four-nitrocatechol inhibits the consumption of oxygen and thereby the DesB reaction. Though this method can provide insight to studying ferrous-dependent dioxygenase enzymes, it can be applied to other systems, such as other metalloenzymes. Don't forget that working within a glove box can be challenging, so take care not to rush while performing this technique.
