Several methods have been used for the estimation of mitochondrial membrane potential (A>|>) which are based on measurement of the distribution of a lipophylic cation across the inner membrane. These include tetraphenylphosphonium ion (TPP) and related compounds. More recently, the methyl (TMRM) and ethyl (TMRE) esters of tetramethylrhodamine have been employed to measure Ay in intact cells based on the measured fluorescence distribution. We found that Ay-dependent accumulation of TMRM and TMRE by isolated rat heart mitochondria caused fluorescence quenching and an approximate 8 nm red shift in the peak excitation and emission wavelengths. Since the accumulation of the free dye by mitochondria should be proportional to ay, we explored the use of a fluorescence ratio method to estimate dye distribution and Ay of isolated mitochondria. Accumulation of TMRM and TMRE by mitochondria was greater than was predicted based solely upon a Nemst distribution. The accumulation was due to non-specific binding, the magnitude of which was estimated as an inner (Ki) and outer (Ko) first order partition coefficient. These coefficients were determined using a valinomycininduced K diffusion potential. The degree of non-specific binding was in the order of TMRE > R123 > TMRM > TPP. After equilibration of the indicator, knowledge of the media dye concentration and the partition coefficients allowed for the calculation of the free matrix concentration, AH, and for calibration of the fluorescence ratio signal. This method is relatively simple and applicable to intact cells.
|Original language||English (US)|
|State||Published - Dec 1 1996|
All Science Journal Classification (ASJC) codes
- Molecular Biology