A. Fedorov

Activity, conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles

The adsorption of a recombinant cutinase from Fusarium solani pisi onto the surface of 100 nm diameter poly(methyl methacrylate) (PMMA) latex particles was evaluated. Adsorption of cutinase is a fast process since more than 70% of protein molecules are adsorbed onto PMMA at time zero of experiment, irrespective of the tested conditions. A Langmuir-type model fitted both protein and enzyme activity isotherms at 25 degrees C. Gamma(max) increased from 1.1 to 1.7 mg m(-2) and U(max) increased from 365 to 982 U m(-2) as the pH was raised from 4.5 to 9.2, respectively. A decrease (up to 50%) in specific activity retention was observed at acidic pH values (pH 4.5 and 5.2) while almost no inactivation (eta(act) congruent with 87-94%) was detected upon adsorption at pH 7.0 and 9.2. Concomitantly, far-UV circular dichroism (CD) spectra evidenced a reduction in the alpha-helical content of adsorbed protein at acidic pH values while at neutral and alkaline pH the secondary structure of adsorbed cutinase was similar to that of native protein. Fluorescence anisotropy decays showed the release of some constraints to the local motion of the Trp69 upon protein adsorption at pH 8.0, probably due to the disruption of the tryptophan-alanine hydrogen bond when the tryptophan interacts with the PMMA surface. Structural data associated with activity measurements at pH 7.0 and 9.2 showed that cutinase adsorbs onto PMMA particles in an end-on orientation with active site exposed to solvent and full integrity of cutinase secondary structure. Hydrophobic interactions are likely the major contribution to the adsorption mechanism at neutral and alkaline pH values, and a higher amount of protein is adsorbed to PMMA particles with increasing temperature at pH 9.2. The maximum adsorption increased from 88 to 140 mg cutinase per g PMMA with temperature raising from 25 to 50 degrees C, at pH 9.2.

Excitation energy transfer and spatial exciton confinement in polyfluorene blends for application in light-emitting diodes

We present picosecond time-resolved studies of thin films of two polyfluorene blends, combining a relatively high energy-gap poly(9,9-dioctylfluorene), PFO, and a polyfluorene derivative with a smaller gap. These studies show both efficient energy transfer from the donor to the acceptor, as well as spatial confinement of the excitons in the acceptor polymers. The optoelectronic characteristics of light-emitting diodes fabricated with such blends reflect the contribution of these two processes. The electroluminescence is almost exclusively from the acceptor polyfluorenes and we observe a significant improvement of the electroluminescence efficiency of the light-emitting diodes fabricated with such blends in comparison to those based on neat films of the polyfluorenes.

Fluorescence of the Single Tryptophan of Cutinase: Temperature and pH Effect on Protein Conformation and Dynamics {

The cutinase from Fusarium solani pisi is an enzyme with a single l‐tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala–Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge–transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10‐fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling‐in‐cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein‐melting temperature and to protein‐backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.

Fluorescence quenching of pyrene monomer and excimer by CH3I

Abstract The fluorescence quenching rate constants of pyrene monomer and excimer by CH 3 I were obtained at several temperatures in methylcyclohexane. Both quenching processes are kinetically controlled, allowing insight on the mechanism of quenching. The rate constants have both temperature-independent and temperature-dependent components. The temperature-independent component for both monomer and excimer fluorescence is consistent with quenching due to enhanced intersystem crossing to a lower energy triplet state. The monomer temperature-dependent component comes from the enhancement of the intersystem crossing to a higher energy triplet state. The thermally activated excimer quenching is associated with the excimer dissociation step to give a pyrene in a second triplet state plus a ground state pyrene molecule.

The influence of nanoparticle architecture on latex film formation and healing properties

We present a study of chain interdiffusion in films formed by specially architectured PBMA nanoparticles by Förster Resonance Energy Transfer -FRET. Polymer nanoparticles contained linear chains with narrower molecular weight distributions than other previous reports, allowing a more detailed study. Apparent fractions of mixing and diffusion coefficients, determined from the quantum efficiency of energy transfer, were used to characterize the interdiffusion mechanism in the different films. The resistance of the films to dissolution by a good solvent was finally correlated with the interdiffusion results, in order to get information about film healing. We concluded that whenever interdiffusion occurs between nanoparticles containing linear chains and fully cross-linked nanoparticles, healing becomes more effective in spite of showing slower interdiffusion. We also observed that particles with longer chains are more effective for film healing. Finally, we concluded that interdiffusion occurs both ways across interfaces in blends formed by particles swollen with linear chains of different molecular weights.

Aggregation of 1,12-bis(1-pyrenyl) dodecane in sol-gel systems

Abstract Silica sol-gel systems were doped with a low concentration of 1,12-bis(1-pyrenyl)dodecane at two pH values. Fluorescence spectra of deaerated samples were measured at regular intervals, far beyond the gel point. At least two intramolecular pyrene dimers and two excimer-like bands ( E 1 and E 2 ) were observed for pH 1.2. In contrast, at pH 2., only one pyrene dimer and the sandwich excimer E 2 were detected. Decay curves of degassed aged gels showed that none of the excimers has a rise time at pH 1.2, but a rise-time around 8 ns was observed at pH 2.5. These results were interpreted in terms of the encapsulation of the probe in primary silica particles, whose structure and size are pH dependent.