The behavior of a fast-responding barbituric acid potential-sensitive molecular probe in bovine heart submitochondrial particles

Abstract

The barbituric acid probe diBa-C2-(5) responds to the formation of a membrane potential (delta psi) in bovine heart submitochondrial particles (SMP) by a CCCP-reversible, 5-7 nm red shift of the probe absorption spectrum. This shift can be enhanced by the addition of nigericin, an observation that indicates that the probe is specifically sensitive to delta psi. Probe-SMP binding analyses indicate that, relative to the resting state, the ratio of the dye dissociation constant to the maximum number of binding sites decreases by a factor of 30 when delta psi is generated. This observation suggests that the origin of the potential-dependent shift of the probe absorption spectrum is increased occupancy of the SMP membrane by diBa-C2-(5). The time course of the ATP-induced diBa-C2-(5) spectral shift in SMP was complete in nominally 0.2 s and could be described by a single-exponential rate equation. There was no evidence for a slower-phase signal when the data collection time period was increased to 250 s. The apparent first-order rate constants obtained from the single exponential analyses of the barbituric acid ATP-generated signal, however, were a linear function of probe concentration at fixed SMP membrane concentration. The resulting second-order rate constant obtained by linear regression was nominally 1 x 10(7) M (dye)-1 s-1; this value is two to three orders of magnitude higher than that of a number of other well-established probes of delta psi in mitochondrial preparations. Based on the invariance of the kinetics of the oxidation of cytochromes c and c1 by ATP-driven reversed electron transport in the presence and absence of the probe, diBa-C2-(5) does not appear to permeate the SMP membrane on a time scale of milliseconds to several minutes.