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In this work, conditions of Suzuki condensation and polymer modification of polymers of benzene and fluorene units have been studied. A new polymer was prepared with statistical equimolecular amounts of two 2,7-dibromo-9,9-bis(6‘-susbtituted-hexyl)fluorene as starting monomers. The C9 position of the poly[9,9-bis(6‘-bromohexylfluoren-2,7-diyl)-alt-co-(benzen-1,4-diyl has been substituted yielding poly[9,9-bis(6‘-cyanohexylfluoren-2,7-diyl)-alt-co-(benzen-1,4-diyl)] with a high degree of conversion. All polymers were completely soluble in THF and chloroform and were characterized by NMR, FTIR, and elemental analysis. Molecular weights measured by gel permeation chromatography coupled with light scattering detector results in polydispersity values between 3.3 and 1.2. Their glass transitions temperature were over 90 and 99 °C by DSC. Comparative studies of optical properties in solution and solid state show similar values indicating that the remote substitution of functional group maintains the spectroscopic characteristics of the backbone. Cyclic voltammetry studies and preliminary results in bias potentials of electroluminiscent devices reveal that these polymers may be promising candidates to be used as hole and electron transporting materials.

The strand-displacement mechanism of Bacillus subtilis phage phi29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, phi29 SSB, to be replicated in vivo. To understand the characteristics of phi29 SSB-ssDNA complex that could explain the requirement of phi29 SSB, we have (i) determined the hydrodynamic behavior of phi29 SSB in solution and (ii) monitored the effect of complex formation on phi29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 +/- 0.1) x 10(8) gs(-1), and its rotational correlation time, 7.0 +/- 0.5 ns, phi29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that phi29 SSB is highly organized within a wide range of temperatures (15 to 50 degrees C), being mainly constituted by beta-sheet elements (approximately 50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of phi29 SSB.

The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage phi29 is absolutely required for viral DNA replication in vivo. About approximately 95% of the intrinsic tyrosine fluorescence of phi29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the phi29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. phi29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the phi29 SSB fluorescence emission (phiF = 0.067 +/- 0.010), (ii) the fast motions of the tyrosine side chains (phi(short) = 0.14 +/- 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I- indicates that phi29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and omega (cooperativity) defining phi29 SSB-poly(dT) interaction, the effective DNA binding constant, K(eff) = K omega, being largely independent of temperature. Altogether, the fluorescent properties of phi29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA.

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-[(4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxyethyl)-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.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 SDP, 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 (?DPH, 36°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 lipidprotein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25° to 36°C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36°-40°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 (~2%) of the order parameter of CE-DPH for Ca2+ concentrations in the millimolar range.

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 lifetimes, 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°C an important fraction of the lipids of the plasma membrane are apparently in the form of solid-like domains (20% at 20°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°C described by Gordon et al. (1983) in a spin-probe study of the platelet plasma membrane.