Talk:Magnesium Green

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Use of Magnesium Green 5N hexapotassium salt to measure ADP-ATP exchange rates

Publications in the MiPMap
Chinopoulos C (2013) Use of Magnesium Green 5N hexapotassium salt to measure ADP-ATP exchange rates. Mitochondr Physiol Network 2013-02-14.

»

Chinopoulos C (2013) MiPNet

Abstract: Edited by Fasching M and Gnaiger E.


O2k-Network Lab: HU Budapest Chinopoulos C


Labels: MiParea: Respiration, Instruments;methods 



Preparation: Isolated mitochondria 

Regulation: ATP, ADP 


HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 


Application of technique:  Online fluorescence measurement of ADP-ATP exchange

[Mg2+]f determination from Magnesium Green (MgG) fluorescence in the extramitochondrial volume of isolated mitochondria and conversion to ADP-ATP exchange rate: Add mitochondria to 2 ml of an incubation medium of your choice. Including the adenylate kinase inhibitor Ap5A into the medium is essential; Mg2+, which is present in the assay medium, activates adenylate kinase. Ap5A is a potent inhibitor of adenylate kinase. MgG fluorescence can be recorded in a fluorometer, using 506 nm and 530 nm excitation and emission wavelengths, respectively. MgG exhibits an extremely high quantum yield (EM[MgG]=75,000 M-1*cm-1). At the end of each experiment, minimum fluorescence (Fmin) can be measured after addition of 4 mM EDTA, followed by the recording of maximum fluorescence (Fmax) elicited by addition of 20 mM MgCl2. Free Mg2+ concentration, [Mg2+]f, is calculated from the equation: [Mg2+]f = (Kd(F-Fmin)/(Fmax-F))-0.055 mM, assuming a Kd of 0.9 mM for the MgG-Mg2+ complex. The correction term -0.055 mM is empirical, and possibly reflects chelation of other ions by EDTA that have an affinity for MgG, and alter its fluorescence. This term is needed to obtain a reliable Mg2+ estimate, as determined from calibration experiments using solutions with known, stepwise increasing, Mg2+ concentrations. ADP-ATP exchange rate is estimated using the recently described method by our laboratory [1], exploiting the differential affinity of ADP and ATP to Mg2+. The rate of ATP appearing in the medium following addition of ADP to energized mitochondria (or vice versa in case of de-energized mitochondria), is calculated from the measured rate of change in free extramitochondrial [Mg2+]f using standard binding equations. The assay is designed such that the ANT is the sole mediator of changes in [Mg2+]f in the extramitochondrial volume, as a result of ADP-ATP exchange [1]. For the calculation of [ATP] or [ADP] from [Mg2+]f, the apparent Kd values should be estimated as described in [1].


Validation of the technique

Mg green checkpoints.jpg
In order be sure that the calibrated MgG signal is converted correctly to ADP-ATP exchange rate, the following 4 check-points should be sought:
  1. After calibration of the MgG signal to [Mg2+]f, the initial [Mg2+]f value must be equal to that added in the cuvette by the experimenter; (i.e. did you add 1 mM MgCl2 in the cuvette? Then your calibrated MgG signal must start from 1 mM free Mg2+).
  2. After addition of ADP (or ATP) to the medium, the [Mg2+]f value must drop to the expected level, after estimating the Kd of ADP (or ATP) for Mg2+; (i.e. if you estimated Kd of ADP for Mg2+ as 0.906 mM, the addition of 2 mM ADP to 1 mM Mg2+ leads to a [Mg2+]f of 0.3939 mM).
  3. After conversion on the calibrated [Mg2+]f to ADP, ATP level must start from “0”, if no ATP was already present in the medium, prior to adding ADP to mitochondria.
  4. The ADP-ATP exchange rate must be completely sensitive to carboxyatractyloside, a specific blocker of the ANT.



[1] Chinopoulos C, Vajda S, Csanady L, Mandi M, Mathe K, Adam-Vizi V (2009) A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT. Biophys J 96:2490-2504.


A kinetic assay of mitochondrial ADP-ATP exchange rate mediated by the adenine nucleotide translocase

» Manual by Christos Chinopoulos: "Follow the links appearing on the left, starting from the top. The 'core' of the method is outlined in the Buffers, chemicals, Kds determination of ADP, ATP to Mg2+, and Conversion of [Mg2+]free to [ATP]. However, it is advisable to go through all of the links, if you wish to understand what you will be actually doing."
  • For ion binding constants of the ATP-system as a function of pH, temperature, and ion composition, see: Gnaiger E, Wyss M (1994) Chemical forces in the cell: Calculation for the ATP system. In: What is Controlling Life? (Gnaiger E, Gellerich FN, Wyss M, eds) Modern Trends in BioThermoKinetics 3. Innsbruck Univ Press:207-12. - »Bioblast link«


MgG Signal and output

  1. Signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
  2. Output: The focus of the output with Magnesium Green is on Type B: Flow, flux, rate.


Mg green concentrations

used by OROBOROS for testing: stock: 2 mM in chamber: 2 µM

Mg2+ calibration experiment

The following calibration experiment was performed with a medium contaminated with Ca2+. Ca2+ impurities are a particular problem because a.) Mg green binds stronger to Ca2+ than to Mg2+ and b.) Ca2+ impurities are very common. The Ca2+ impurities were complexed by an EGTA titration before the Mg2+ calibration, without (in this case) negative effects on the Mg2+ performance.

Titrations:

  • Mg green 2 µL 2 mM --> 2 µM
  • EGTA titration: 3 * (5 µl 20 mM --> 50 µM) until very small changes in signal
  • DTPA 2 µl 20 mM -->20 µM
  • 10 * ( 4 µl MgCl2 0.1 M --> 0.2 mM)
  • EDTA 16 µl 500 mM --> 4 mM
  • EDTA 2 µl 500 mM --> 0.5 mM: no further change
  • MgCl2 80 µl 1 M --> 40 mM
  • MgCl2 20 µl 1 M --> 10 mM: no further change


(To allow performance tests a substance mimicking light scattering of typical samples was added to the medium, however this is not relevant for the results presented here.)

The addition of a very small concentration of either EDTA or DTPA in the beginning of the experiment is suggested by the produced of the fluorophores (for all similar fluorophores) to remove traces of transition metal cations.


Shown in the DatLab graph is the original raw voltage as recoded by the fluorescence sensor and the Mg2+ concentration. For calibration determining KD and slope) and plotting the calculated Mg concentration (via the experimental scripting function of DatLab) the following relation was used

[Mg2+] =  Kd * ( F- Fmin) / (Fmac -F) + intercept

with

F .......Fluorescence signal Fmin..... minimal fluorescence signal, free Mg2+ concentration = 0, established by excess EDTA Fmax .....maximal fluorescence signal, fluorophore indicator saturated with Mg2+, established by excess of MgCl2


MF686b A module DL.png

MF686b A module calibration.png

The determined KD value (1.8 mM) is influenced by the presence of both Ca2+ and EGTA. However, by using the the determined KD value the linearity of the calculated Mg2+ concentration is still very good. Significant concentrations of free Ca2+ have to be avoided to due the strong binding to Mg green. Concentrations of EDTA or DTPA should be kept as small as possible because the will reduce the free Mg2+ concentration to a different degree at low and high Mg2+ concentrations. A medium that is as Ca2+ free possible should be used as a matter of principle, even if this example shows that good results may be be obtained even in the presence of heavy Ca2+ contamination.

In this example all Mg concentration from 0.2 mM to 2 mM as input into the calibration calculation. However, in a real experiment for the calculation of KD only those magnesium concentrations should be used that cover the concentration range observed in the experiment.

Fasching Mario 10:34, 17 March 2015 (CET)


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