EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The overall goal of this procedure is to characterize the redox properties of an enzyme or protein. This is accomplished by first inserting a working and reference electrode connected to a bolt meter to a redox titration vessel above a magnetic stir. The second step is to add protein buffer and a mixture of different redox mediators to the titration vessel.

Next, a chemical reduct and oxidant are used to poise a potential to different values and electron paramagnetic resonance or EPR samples are taken. The final step is to record EPR spectra of the samples and prepare titration curves. The main advantage of this technique of our existing methods, like full telemetry, is that complex proteins with multiple different cofactors can be readily analyzed.

To begin this procedure, connect the silver chloride reference electrode and a platinum wire electrode to a redox titration vessel. Then connect both electrodes to a volt meter that can measure from millivolts up to volts. Insert a small stirring bar and place the titration vessel above a magnetic stirah.

Next, fill the titration vessel with previously prepared buffer B mediator mix solution and protein solution up to two milliliters to give final concentrations of 72 micromolar protein and 38 micromolar of each mediator. After stirring for 30 minutes, switch on the volt meter. Introduce 10 clean quartz EPR tubes in an anaerobic chamber.

Then fill a small liquid nitrogen doer with liquid nitrogen. After waiting until the potential of the solution is stable, note the potential given by the volt meter following this. Draw the first sample of 200 microliters and inject it into an EPR tube.

Cap the tube with a rubber stopper and remove the tube from the anaerobic chamber. Freeze the tube by slowly inserting the tip in the liquid nitrogen. Once a hissing sound is heard, slowly insert the rest of the tube so that the sample part is fully submerged.

Add one to two microliters of a previously prepared two millimolar sodium Tite solution to the solution in the titration vessel to lower the potential of the solution to negative 0.6 volts. After drawing a sample, note the actual potential given by the volt meter to increase the potential by circa 50 millivolts. Add one to two microliters of a previously prepared two millimolar sodium cyanide solution.

To record EPR spectra of the different titration samples, evacuate a quartz cryostat to less than 10 to the minus five bar. Using a high vacuum pump switch on the water cooling device of the EPR magnet, then open the flow of dry air through the EPR cavity. After switching on the power supply to the magnet and the computer of the EPR, run the frequency calibration program.

When finished, set the EPR measurement parameters. Next, connect the liquid helium doer to the cryostat to cool the cavity to nine to 16 Kelvins. Introduce the EPR sample in the cavity, then record the EPR spectrum.

Finally, plot the EPR signal amplitude at a G value that is characteristic for the paramagnetic species of interest against a potential a three iron, four sulfur cluster, a four iron, four sulfur cluster, and a mononuclear iron center were identified as redox active co-factors. In nar one quantification of the EPR signals indicated that the four iron, four sulfur cluster and mononuclear iron are equimolar at approximately 60%of the protein concentration and that the three iron four sulfur cluster content is only 5%The three iron four sulfur cluster is most likely a degradation product and incomplete cluster. The midpoint potentials of the different co-factors were determined to be positive 0.003 volts for the mononuclear iron center.

Couple negative 0.1 25 volts for the three iron four sulfur couple and between negative 0.45 and negative point 50 volts for the four iron four sulfur couple. The low potential of the four iron four sulfur cluster indicates that it does not have a redox roll in the protein as it will be very difficult to reduce the cluster. The mononuclear iron center can be reduced or oxidized in the cytoplasm, making a redox roll of this center possible.

While attempting this procedure, it's important to remember that the titration can be performed in an oxidizing or reducing direction. The best choice is dependent on the stability of the cough factor under different redox states.