Contact: Dr. Christos Chinopoulos MD, PhD
'Under the hood' advices

Under the hood advice #1: Regarding the buffer that you will use to measure ADP-ATP exchange rate ('ANT buffer'), use only 20-30 mM [Cl-] salts, the remaining anion can be gluconate. ~20 mM [Cl-] is within the physiological range of intracellular [Cl-] for all tissues. ANT is inhibited at high [Cl-]; 140 mM [Cl-] inhibits ANT activity by ~35%, while with 140 mM gluconate inhibition is only ~7% (Gropp et al., 1999). Furthermore, mitochondria exhibit similar respiration rates and membrane potential but significantly lower steady-state rates of ROS formation in 20 mM [Cl-], as compared to mitochondria exposed to 140 mM [Cl-].

Under the hood advice #2: Use minimum 5 mM, preferably 10 mM [Pi] in your buffer, for the following reasons: There are two Pi carrier isoforms in mitochondria with Km values for Pi (on the external membrane surface) of 2.2 and 0.78 mM, respectively (Palmieri, 2004). The high concentration of phosphate (10 mM) achieves 81.97 % and 92.76 % saturation of the low and high-affinity Pi carrier, respectively. At lower concentrations extramitochondrial Pi becomes rate-limiting for oxidative phosphorylation (Tager et al., 1983), while at saturating [Pi] the rate of Pi transport greatly exceeds the net rate of ATP synthesis (Ligeti et al., 1985). At [Pi] ≤ 3 mM ATP formation is largely dependent on the F0-F1-ATP synthase. Finally, Pi has been shown by mathematical modeling to play a significant role in stimulating both oxidative phosphorylation and tricarboxylic acid cycle (Wu et al., 2007), as originally proposed by Bose (Bose et al., 2003). 10 mM KH2PO4 does not affect free [Mg2+] measured by MgG. This is partly because at pH=7.25, KH2PO4 dissociates to H2PO4- and HPO42- (pK2=7.21) and neither of these ions form a precipitation with Mg2+ (PO43- is the only component that forms Mg3(PO4)2 precipitate, but this ion is essentially not present at this pH (pK3=12)). On the other hand, Mg2+ also forms soluble coordination compounds with phosphate anions (preferably with HPO42-). However, chelation of Mg2+ by HPO42- in the low millimolar range is negligible, since the dissociation constant for the [Mg2+-HPO42-] complex is 210 mM (Dawson et al., 1986), although a much lower value has been reported elsewhere (Wu et al., 2007).

Under the hood advice #3: Include BSA in your buffer. It foams, so don't shake. The importance of including bovine serum albumin in the assay medium is two-fold: firstly, albumin binds both fatty acids and their coenzyme A esters, that can inhibit ANT activity (Wojtczak and Zaluska, 1967), (Morel et al., 1974); secondly, fatty acids exhibit uncoupling properties plus inhibitory effects on respiratory chain components (Wojtczak and Schonfeld, 1993) that decrease pmf, particularly with succinate as a substrate (Tretter et al., 2007).

Under the hood advice #4: Don’t use less than 1 mM Mg2+ in your buffers if you investigate isolated mitochondria, and 1.5 mM Mg2+ if you investigate permeabilized cells. The use of 1 mM total [Mg2+] yields 0.32-0.6 mM free [Mg2+] (depending on the type and amount of adenine nucleotide present in the extramitochondrial medium), which is within the physiological range of cytosolic free [Mg2+] (Rodriguez-Zavala and Moreno-Sanchez, 1998), (Rutter et al., 1990), (Jung et al., 1997).

Under the hood advice #5: Don't add less than 1 mM ADP to initiate ADP-ATP exchange (best combination: 2 mM ADP and 1 mM MgCl2 for isolated mitochondria, and 2 mM ADP and 1.5 mM mM MgCl2 for permeabilized cells).

Under the hood advice #6: Include Ap5A (25-50 mM) in your buffer.

Under the hood advice #7: ATP and ADP stocks: i) use of highest purity available; ii) titrate to pH = 6.2 - 6.9 (with KOH or NaOH), do NOT titrate to pH 7.04-7.4;  iii) NEVER use Mg2+ salts!!!; iv) keep in aliquots.

Under the hood advice #8: Estimate how many experiments you will perform, and prepare the required amount of buffer prior to the experiments; add to it 1.1 mM MgG, and keep it at 37 0C (or the temperature of your choice). This will eliminate the potential differences in MgG concentration from one experiment to another, plus minimize signal drifts.