Phosphorescence quenching method for measurement of intracellular Po-2 in isolated skeletal muscle fibers

Values of skeletal muscle intracellular Po, during conditions ranging from rest to maximal metabolic rates have been difficult to quantify. A method f or measurement of intracellular Po-2 in isolated single skeletal muscle fib ers by using O-2-dependent quenching of a phosphorescent-O-2 probe is descr ibed. Intact single skeletal muscle fibers from Xenopus laevis were dissect ed from the lumbrical muscle and mounted in a glass chamber containing Ring er solution at 20 degrees C. The chamber was placed on the stage of an inve rted microscope configured for epi-illumination. A solution containing pall adium-mesotetra (4-carboxyphenyl) porphine bound to bovine serum albumin wa s injected into single fibers by micropipette pressure injection. Phosphore scence-decay curves (average of 10 rapid flashes) were recorded every 7 s f rom single cells (n = 24) in which respiration had been eliminated with NaC N, while the Po-2 of the Ringer solution surrounding the cell was varied fr om 0 to 159 Torr. For each measurement, the phosphorescence lifetime was ca lculated at the varied extracellular Po-2 by obtaining a best-fit estimate by using a monoexponential function. The phosphorescence lifetime varied fr om 40 to 70 mu s at an extracellular Po-2, of 159 Torr to 650-700 ps at 0 T orr. The phosphorescent lifetimes for the varied Po-2 were used to calculat e, by using the Stern-Volmer relationship, the phosphorescence-quenching co nstant (100 Torr(-1) s(-1)), and the phosphorescence lifetime in a zero-O-2 environment (690 mu s) for the phosphor within the intracellular environme nt. This technique demonstrates a novel method for determining intracellula r Po-2, in isolated single skeletal muscle fibers.