Fluorescence changes on contractile activation in TnC(DANZ) labeled skinned rabbit psoas fibers

The increase in fluorescence of dansylaziridine (DANZ) labeled troponin C (TnC(DANZ) substituted into skinned rabbit psoas fibers was determined as a function of the pCa. The fluorescence data are expressed as the ratio of two wavelength bands, one that sees the fluorescence of TnC(DANZ), and one that sees background fluorescence and scatter. The percent TnC replaced with TnC(DANZ) was varied between 10 and 50% and, the fibers were randomly stretched, at the start of each experiment, between 10 and 50%. A large ratio increase accompanies increase in [Ca2+]. The pCa/force data are best fit by the Hill equation but the pCa/ratio data are best fit by a model in which Ca2+ binds in two phases. The position of the force curve on the pCa axis varies little between fibers, in contrast to that of the ratio or A-fluorescence curve. In accord with previous reports the delta-fluorescence can be left of the force on the pCa axis (type I) or superimpose in part on the force (type II). Not described previously, we find curves in which the second phase of the ratio cross-over the pCa/force curve. This type III relationship is found only in fibers less than 3 weeks postmuscle harvest. We propose that the first, relatively invariant, phase of the biphasic pCa/ratio curve accompanies Ca2+ binding to either of the two low affinity sites on TnC(DANZ) as it does for TnC in solution. The second, highly cooperative, phase of the ratio curve that accompanies muscle contraction and enhanced Ca2+ binding is initiated when sufficient Ca2+ is bound to overcome inhibitory systems. Loose coupling between the initial Ca2+ binding and the cooperative switch point may account for much of the variation in the shape and position of the pCa/ratio curve. There is evidence that, in the overlap zone, weakly attached myosin cross-bridges enhance cooperation between the regulatory units of the thin filaments.