An evaluation of the effects of five buffers on respiratory parameters of isolated mitochondria

Abstract

Certain compounds, recently introduced as buffers for biological work in the pH range 6–8, were evaluated by measurement of their effects on mitochondrial respiration. Respiratory control, respiration rate, ADP/O ratio, and the effect of added cytochrome c were measured on bean cotyledon mitochondria, after isolation and incubation of the mitochondria in one of five buffers: phosphate, Tris, Tricine (N-tris(hydroxymethyl)methylglycine), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), and HEPES (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid). In all parameters measured, except response to added cytochrome c, purified TES was found to be superior to all other buffers. HEPES was the best in prevention of leakage of cytochrome c from the mitochondria but in other respects was similar to Tricine. Phosphate buffer proved superior to Tricine only with respect to cytochrome c retention by the mitochondria. Tris was the poorest buffer in which to measure any of these parameters. Aging the mitochondria in the various buffers amplified the inadequacies of the buffers and served to show the superiority of purified TES.