Determination of the cardiolipin content of individual mitochondria by capillary electrophoresis with laser-induced fluorescence detection

Abstract

We report the application of capillary electrophoresis (CE) with postcolumn laser-induced fluorescence (LIF) detection to measure the cardiolipin content of individual mitochondria from cultured NS1 cells. Mitochondria were isolated by differential centrifugation and stained with the fluorescent dye 10-N-nonyl acridine orange which stoichiometrically binds to cardiolipin in a 1:1 or 2:1 ratio depending on the dye concentration. The green fluorescence resulting from the 1:1 complex was chosen for analysis because it is substantially more intense than the red fluorescence resulting from the 2:1 complex. Two dye concentrations that resulted in maximal and submaximal formation of the 1:1 10-N-nonyl acridine orange-cardiolipin complex were identified by spectrofluorometry. Individual mitochondria stained with both dye concentrations were separated and detected by CE with LIF detection. The data from mitochondria dosed with the lower dye concentration, where it is assumed that all the dye added to the mitochondrial sample was bound to cardiolipin, were used to derive a sensitivity factor relating fluorescence intensity of a mitochondrial event to its cardiolipin content. Using this factor, the cardiolipin contents of individual mitochondria stained with the higher dye concentration were determined, and ranged from 1.2 to 920 amol, with a median value of 4 amol. These results suggest a new strategy for estimating the organellar content of compounds that can be fluorescently tagged.

Mitochondrial DNA Damage in Iron Overload

“…The cells were suspended in this isolation buffer to 8 ϫ 10 6 cells/ml and disrupted by nitrogen cavitation at 4°C using an ice-cooled cell disruption bomb (Parr Instrument Co., Moline, IL). Total cell lysates were used directly for carbonyl assays and mitochondria were prepared by differential centrifugation using a modification of the procedure described by Fuller et al (11). Following removal of large debris by centrifugation at 500 ϫ g for 10 min at 4°C, the supernatants were centrifuged at 14,000 ϫ g for 20 min at 4°C to obtain mitochondrial pellets.…”

Oxygen Tolerance and Coupling of Mitochondrial Electron Transport

“…Estimation of Mitochondrial Protein Carbonyls-Mitochondria were prepared by differential centrifugation using a modification of the procedure described by Fuller et al (27). Briefly, cells grown on 10-cm dishes were washed twice with cold mitochondrial isolation buffer (250 mM sucrose, 10 mM Tris-HCl, pH 7.8, 1 mM EDTA, pH 8.0).…”

Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations

“…For organelles at biological pH, the electrophoretic mobility is negative because a net negative electrical charge accumulates on the surface organelle as a result of the contributions of ionized phospholipids, proteins, and glycoproteins. Thus, organelles that are kept at biological pH tend to migrate towards the anode in the presence of an electric field when other factors such as electroosmotic flow are suppressed [20][21][22][23][24]. In contrast to the electrical surface charge, morphology and size do not seem to have a strong effect on the electrophoretic mobility of an organelle.…”

“…Recently, we reported the use of capillary electrophoresis to separate organelles including mitochondria [22][23][24], acidic organelles [33], and nuclei [34]. Using this technique, about 1 nl of organelle suspension is sampled electrokinetically or hydrodynamically into a separation capillary (30-40 cm long, 50 m internal diameter).…”

“…This detector has been used to measure individual organelle electrophoretic mobilities, count mitochondria, and estimate the cardiolipin content of individual organelles. Worthy noting in some of these reports [22][23][24] was that the distributions resulting from individual mitochondrial electrophoretic mobility measurements varied even when using the same experimental approach and separation conditions. Although some of these variations in electrophoretic mobility may be attributed to the variation in the mitochondrial source, we hypothesized that the isolation or preservation procedures for mitochondria may have an impact on the electrophoretic mobilities of these organelles.…”