This protocol highlights the role of sodium as a second messenger and also shows the existence of partially differentiated ubiquinol pools. This technique demonstrates an extremely simple approach for the study of ubiquinol pools which otherwise requires very specific cell models. This approach may be used for the study of electron transfer in other membranes, particularly of enzymes within lipid drops.
Split the samples into four sub-samples of 20 micrograms each and label them A to D.Split samples A and B into two sub-samples of 10 micrograms each and label them as A1, A2, B1, and B2.Prepare C1/C2 buffer as described in the text protocol and preheat to 37 degrees Celsius. In a one milliliter cuvette, add each of the sub-samples to 30 microliters of cytochrome c and 10 microliters of malonate. Add C1/C2 buffer to bring up the volume to 980 microliters in cuvettes A1 and B1 and 979 microliters in A2 and B2.Add 10 microliters of one-molar potassium chloride in cuvettes A1 and A2 and 10 microliters of one-molar sodium chloride in B1 and B2.Add one microliter of one-millimolar rotenone into the cuvettes A2 and B2.Right before the measurement, add 10 microliters of 10-millimolar NADH into all cuvettes.
Flip the cuvette three times and place it in the spectrophotometer. An the attached software, click on Measure then Parameters, then go to General and set the measurement parameters at wavelength of 550 nanometers and time at four minutes of reading. Click on OK and Start to begin the experiment.
At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As.Split samples C and D into two sub-samples of 10 micrograms each and label them as C1, C2, D1, and D2.Mix each of the sub-samples in a one-milliliter cuvette with 30 microliters of cytochrome c and one microliter of rotenone. Add C1/C2 buffer preheated at 37 degrees Celsius to bring up the volume to 980 microliters in cuvettes C1 and D1 and 970 microliters in C2 and D2.Add 10 microliters of one-molar potassium chloride in cuvettes C1 and C2 and 10 microliters of one-molar sodium chloride in D1 and D2.Add one microliter of one-millimolar antimycin A into the cuvettes C2 and D2 right before the measurement and 10 microliters of one-molar succinate into all cuvettes. Flip the cuvette three times and place it in the spectrophotometer.
Perform the measurement and save the slope comprising the linear increase of absorbance at the end of the measurement. This protocol was used to study the effect of sodium ions on the reduction of cytochrome c by mitochondrial membranes upon NADH or succinate addition. Absorbance traces generated at 550 nanometers for samples A and B were corrected for their corresponding inhibition.
These traces showed a similar slope, indicating similar NADH-cytochrome c oxidoreductase activity. However, traces for samples C and D were different, showing that succinate-cytochrome c oxidoreductase activity of sample C is higher than that of sample D.In order to normalize the amount of change measured with this technique, other methods can be further performed, such as isolated complex I activity, complex II, or complex III. Using this technique, we are exploring the physiological settings in which sodium may be involved as a second messenger.
