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The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A.

We have investigated the complex behaviour of the time resolved fluorescence intensity and anisotropy of trans-parinaric acid, incorporated into fragments of the plasma membrane of human platelets and in multibilayers of lipids extracted from that membrane. It is shown that the observation of anisotropies that increase at long times can be satisfactorily interpreted by assuming two populations of the fluorescence probe with distinct life-times, rotational relaxation times and order parameters. The heterogeneous probe distribution was correlated with a similar heterogeneity in the lipid composition of the bilayer, modulated by temperature. Below 35 degrees C an important fraction of the lipids of the plasma membrane are apparently in the form of solid-like domains (20% at 20 degrees C). However, in the physiological temperature range that solid/fluid heterogeneity is almost negligible. Since these effects were also observed in multibilayers of lipids from the platelet membrane, the formation of solid-like clusters appears to arise from lipid-lipid interactions only, and most probably involving cholesterol. These results support the previous finding of a lateral phase separation for temperatures less than 37 degrees C described by Gordon et al. (1983) in a spin-probe study of the platelet plasma membrane.

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-[(4-trimethyl- amino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxy- ethyl)-1,6-diphenyl-1,3,5-hexatriene, 16(9-anthroyloxy)-palmitic acid, CIS-parinaric acid, trans-parinaric acid and perylene) to report changes induced by temperature and Ca2+ in the plasma membrane of human platelets has been examined. The steady-state fluorescence anisotropy of the probes was compared after being incorporated into whole resting platelets, fragments of platelet plasma membrane and multilayers of lipids extracted from these membranes. In addition, we have investigated the molecular order and dynamics of the three preparations by time-resolved fluorescence depolarization of DPH and CE-DPH as a function of temperature and Ca2+ concentration. The high values of the order parameters found in intact platelets (SDPH, 36 degrees C = 0.70) were almost identical to those in membrane fragments and lipid vesicles, suggesting that lipid-lipid interactions and, therefore, the lipid composition are the main factors influencing the probe order parameter. Other lipid interactions such as those with membrane proteins and intracellular components have little effect on the SDPH in platelets. These measurements also showed that the stationary fluorescence anisotropy of DPH and CE-DPH in platelets is largely determined (80%) by the structural order of the lipid bilayer. Therefore, the previous "microviscosity" values based on stationary anisotropy data reflect the alignment and packing rather than the mobility of the bilayer components. The dynamic component of the anisotropy decay of these probes was analyzed in terms of the wobbling-in-cone model, allowing an estimation of the apparent viscosity of platelet plasma membrane (eta DPH, 36 degrees C = 0.5 P) that is similar to that of the erythrocyte membrane. This value decreased substantially in multilayers of native lipids, indicating a large effect of the lipid-protein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25 degrees to 36 degrees C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36 degrees-40 degrees C interval. This may be related to the end-point of the lipid phase separation reported by Gordon et al. (1983). Finally, the rigidifying (lipid ordering) effect of Ca2+ on the platelet plasma membrane could also be observed by the fluorescence anisotropy measurements, in the form of an increase (approximately 2%) of the order parameter of CE-DPH for Ca2+ concentrations in the millimolar range.