Step 4: Continuing in same cells in the chamber, add 0.25 mM ADP in subsequent steps for a total of 19 steps (blue trace, about 50 sec of recording time per addition). This is shown in figure above, on the right side of the dashed line. Likewise, in a new cell preparation, repeat Steps 1- 4, but with 11 additions of 0.2 mM ATP instead of ADP, also shown in the figure above (orange trace).
Step 5: Convert the MgG signal of both left and right part of the figure shown above to free [Mg2+]. To do this, use this excel (you can see a screenshot of the excel sheet below, followed by instructions).
The method relies on the quantitative determination of free [Mg2+] in the extramitochondrial medium. Thus, MgG raw fluorescence needs to be calibrated to free [Mg2+] (see step 5). Furthermore, the affinity constants of ADP for Mg2+ and ATP for Mg2+ for the pertaining conditions (composition of the medium, temperature, ionic strength, type of biological material, planetary alignment, etc), must be determined.
Below, an example is presented for calibrating MgG fluorescence to [Mg2+] and determining the affinity constants of ADP for Mg2+ and ATP for Mg2+ (Kd_ADP and Kd_ATP, respectively) for permeabilized cells, using an Oroboros equipped with an O2k-Fluorescence LED2-Module:
Step 1: Resuspend cells in 2 ml of 'ANT buffer' containing 50 μM AP5A, 5 mM NaF, 0.2 mM BeSO4, 25 μM Na3VO4, 1 μM carboxyatractyloside (cATR), 10 μg/ml oligomycin and 50 μM digitonin and add them to a chamber of an Oroboros Oxygraph-2k. Then, add 1.1 mM MgG to the chamber. MgG fluorescence is recorded by the O2k-Fluorescence LED2-Module at a 1 Hz acquisition rate. Use Filter set 'MgG/CaG', together with 'Fluorescence-Sensor Blue'. If you choose to use a conventional fluorimeter, use 505 nm for excitation and 535 nm for emission. MgG has a very high extinction coefficient (75,000 cm-1*M-1, i.e. it is very bright), so rather use a low excitation light intensity. Experiments are performed at 37 oC, or the temperature of your choice. If you use isolated mitochondria, you don't need to add NaF, BeSO4, Na3VO4, or digitonin. If you use homogenates, you definitely need to add NaF, BeSO4, and Na3VO4, but not digitonin. Wait for signal stabilization.
Step 2: Add 1 μM EGTA (to chelate nominal amount of Ca2+ in the buffer). If you see a drop in MgG fluorescence, add another bolus of 1 μM EGTA. Keep adding boluses of 1 μM EGTA until you see no further drop in MgG fluorescence. Calculate the amount of EGTA that you have cumulatively added. This should be the amount of EGTA that you need to add to all of your buffers in order to minimize Ca2+ contamination. In the same experiment, after the addition of EGTA boluses, add 1 μM EDTA (to chelate nominal amount of Mg2+ in the buffer). If you see a drop in MgG fluorescence, add another bolus of 1 μM EDTA. Keep adding boluses of 1 μM EDTA until you see no further drop in MgG fluorescence. Calculate the amount of EDTA that you have cumulatively added. This should be the amount of EDTA that you need to add to all of your buffers in order to minimize Mg2+ contamination. If you change buffer composition, you need to perform step 2 again in order to determine how much EGTA and EDTA you need to add in order to eliminate contaminations with Ca2+ and Mg2+. Note that it would be a huge mistake to add millimolar of EGTA and EDTA 'just to be sure'. The method relies on the quantitative determination of free [Mg2+] in the extramitochondrial medium, plus, the amount of free Ca2+ will also affect the binding of ADP and ATP to Mg2+, see under 'Affinities of ions to ADP, ATP'.
Step 3: MgG fluorescence signal is recorded upon stepwise additions of 0.1 mM MgCl2 for a total of 10 additions (blue trace, about 50 sec of recording time per addition). This is shown in the figure below, on the left side of the dashed line.
Instructions for using 'CALIBRATION OF MgG RAW FLUORESCENCE TO [Mg2+].xlsx' Note: you can only input data (variables) in yellow-colored boxes. Note: Calculated data appear in green-colored boxes. The excel sheet is populated with typical data; please replace them with your own. First: In column J, input the time scale of your MgG traces (in seconds), assuming that the experiments shown in the blue and orange traces were performed simultaneously. If you performed them sequentially, please be exact and make the additions at nearly the exact same times so that you can use the same time scale for both experiments. Second: In column K, input the MgG raw fluorescence data obtained from the additions of MgCl2 pulses followed by the additions of ADP pulses, as shown in the blue trace. Third: In column L, input the MgG raw fluorescence data obtained from the additions of MgCl2 pulses followed by the additions of ATP pulses, as shown in the orange trace. Fourth: In column C, input the cumulated amount of MgCl2 added, in mM. Fifth: In colum A, input the steady states of MgG raw fluorescence obtained right after each MgCl2 pulse addition, obtained from the orange trace. Sixth: In colum B, input the steady states of MgG raw fluorescence obtained right after each MgCl2 pulse addition, obtained from the blue trace.
If you have done the above correctly, columns M and N will be populated with the calibrated MgG fluorescence to free [Mg2+]. Column M is the calibrated blue trace (ADP additions after MgCl2 additions), while column N is the calibrated orange trace (ADP additions after MgCl2 additions). Furthermore, in the graph entitled "Calibrated MgG fluorescence to [Mg2+], in mM", the left and right parts of the blue and orange traces must superimpose, and in the graph entitled "2nd order polynomial fit", there should be two dotted curves fitting your data indicating how reliable is the calibration of MgG raw fluorescence to free [Mg2+].
If in the graph entitled "Calibrated MgG fluorescence to [Mg2+], in mM", the left and right parts of the blue and orange traces superimpose, and in the graph entitled "2nd order polynomial fit" the curves 'fit well' to your data (i.e. r2> 0.95), it means that you can calibrate MgG raw fluorescence reliably; you can proceed to step 6 in order to determine the affinity constants of ADP for Mg2+ and ATP for Mg2+ (Kd_ADP and Kd_ATP, respectively), for your experimental buffer and conditions.
Step 6: The determination of the affinity constants of ADP for Mg2+ and ATP for Mg2+ (Kd_ADP and Kd_ATP, respectively) can be done in SigmaPlot. Gergo and myself have tried very hard to produce an excel sheet for this task, but failed to do so. Below I provide instructions for using a SigmaPlot notebook (jnb file, with screenshots) for determining Kd_ADP and Kd_ATP for Mg2+. This notebook has been generated using SigmaPlot version 11.0, build 18.104.22.168. (If you cannot use SigmaPlot, then please donate 5 USD, send me your data, and I will send you the estimated values of both Kd_ADP and Kd_ATP for Mg2+ within 1-2 business days).
Substep I) In the SigmaPlot notebook, input in column 1 the cumulated amount of ADP added, in mM, and in column 2 the calculated [Mg2+] obtained from the steady states right after each addition of ADP pulse, see screenshot below:
Substep II) Next, click on Statistics -> Non-linear Regression -> Regression Wizard (for SigmaPlot 8 and above, press F5 to get directly to step III)
Substep III) Next, click on New...
Substep IV) Next, type the following information into the appropriate dialog windows:
Equation: f=(Mgtotal-x-Kd+sqrt((Mgtotal-x-Kd)^2+4*Kd*Mgtotal))/2 fit f to y
Initial parameters: Kd=0.9 (or whatever you think is a good guess for Kd_ADP) Mgtotal=1.0221 (estimated free Mg2+ before addition of first bolus of ADP or ATP)
Variables: x=col(1) y=col(2)
Options Iterations: 200
Step size 1
Substep V) Next, click OK and give a name for this Equation (whatever you like, let's call it "Monster High" for now)
VI) Next, click OK, then click Monster High and then click Next
Substep VII) Next, make sure that the Variables are correctly selected (x column is [ADP] cumulated amount, y column is calculate free [Mg2+]:
Substep VIII) Click Next, and you finally get the value of Kd_ADP: Kd= 9.143e-1 StdErr 2.818e-2, which is equivalent to Kd=0.9143 mM +/- 0.02818 mM (SEM).
In order to obtain the value of Kd_ATP for Mg2+, repeat substeps I-VIII, but in step I you must input the cumulated amount of ATP added (in mM), and in column 2 the calculated [Mg2+] obtained from the steady states right after each addition of ATP pulse (in mM); also in step IV, in the Initial parameters you must input Kd=0.1 (or whatever you think is a good guess for Kd_ATP).