THE BEHAVIOR OF A FAST-RESPONDING BARBITURIC-ACID POTENTIAL-SENSITIVE MOLECULAR PROBE IN BOVINE HEART SUBMITOCHONDRIAL PARTICLES

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 . 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.