Light-induced axial and radial shrinkage effects and changes of the refractive index in isolated bovine rod outer segments and disc vesicles

Flash-induced transients in the near-infrared scattering of bovine rod outer segments and isolated discs are investigated. Their common characteristic is the saturation at a rhodopsin bleaching of ca. 10%, which was previously described for the so-called signalP. The theory is based on the Rayleigh-Gans-approximation and on a cylindrical particle shape. This treatment is shown to be applicable in the measured angular range (in general30), in spite of the polydisperse shape of the real particles. Using the angular dependence of the relative intensity change (difference scattering curve), changes of the polarizability (refractive index) and of the particle shape can be distinguished. Model difference scattering curves are calculated for the dimensions of the rod outer segments. Static scattering measurements are used for an estimation of the average particle shape: the isolated disc samples appear to contain flat discs as well as an admixture of rod-like structures (ca. 1% of the total scattering mass); in rod outer segment preparations, a contribution of non-rodlike scattering is found which is strongly dependent on the treatment of the sample. The flash induced transients were measured using randomly oriented particles (discs and rod outer segments) and axially oriented rod outer segments. The angular dependence of the amplitude yields its difference scattering curve. On suspensions of isolated discs, which were re-loaded with the proteins extracted at low ionic strength, one single signal is observed (termedP_D, first order,=0.6–1.2 s). Using randomly oriented rod outer segments, a signal with complex millisecond kinetics (termed signalP) and a slow signal (termedP_S, first order,=5–25 s) can be distinguished kinetically. In the axially oriented rod outer segments, theP-signal splits into a fast axial (10 ms) and a slower radial component (50–100 ms). The slow signalP_S observed in ROS and the signalP_D in discs have one common physical interpretation as local changes of the polarizability, directly observed in light-scattering as a change of the refractive index. The fast signalP in ROS, however, has no detectable local component but represents a pure shrinkage effect. On the axially oriented system, this shrinkage turns out to be axial and radial with different kinetics. Only rough estimations for the relative shrinkage effects and refractive index changes can be given. One obtains for 1% rhodopsin bleaching:n/n10^–4,L/L10^–2,R/R5×10^–4. Assuming a fluid plane for the disc membrane, the planar shrinkage induced by one bleached rhodopsin is estimated from the radial shrinkage as ca. 300 å². This high value is discussed in relation to the binding of rhodopsin to the GTP-binding protein which is involved in comparable effects described by Kühn et al. (1981). According to our data, a chemical binding process in milliseconds is only indicated in the isolated disc; in the closed disc stack of the rod outer segment, only weak (fast) local interactions are consistent with the difference scattering data. A turn or lift of the GTPase would better satisfy this condition and explain the above high value for the individual shrinkage effect.Abbreviations ROS rod outer segments - RGA Rayleigh-Gans-approximation