Stanisław Wysocki

Fluorescent molecularly imprinted polymer studied by time-resolved fluorescence spectroscopy

Abstract Molecularly imprinted polymers (MIPs) with template-selective recognition sites and incorporated fluorosensor were prepared against adenosine 3′,5′-cyclic monophosphate (cAMP). The time-resolved fluorescence decay analysis was used to investigate the specificity and affinity of the binding of template molecules to the MIP. The fluorescence decays were modelled in terms of lifetime distributions and two fluorescence lifetimes were observed for the MIPs. The lifetime distributions are interpreted in terms of the heterogeneity of the functionalised imprinted cavities. Quenching of fluorescence of the imprinted polymer with increasing concentration of aqueous cAMP was observed from the fluorescence lifetime parameters data. The mechanisms of interactions between the cAMP and fluorosensor molecules inside the imprinted cavity in comparison with the interactions in solution are discussed.

FLUORESCENCE STUDY OF FUNGAL LIPASE FROM HUMICOLA LANUGINOSA

Abstract Time-resolved and steady-state fluorescence quenching measurements have been performed to study two different conformations of the fungal lipase from Humicola lanuginosa . The intrinsic fluorescence of tryptophan Trp89 residue, located in the ‘lid’ region, has been used as a probe for the dynamics of protein. The native (‘closed-lid’) form of the enzyme has been found to decay as a triple exponential with time constants and relative contributions of 5.4 ns (74.3%), 2.2 ns (20.4%) and 0.4 ns (5.3%). A comparison of recovered decay parameters obtained for native and mutated H. lanuginosa lipase shows that Trp89 contributes about 61% to the class of emitting species with the lifetime of 5.4 ns. The fluorescence quenching data show that three out of four tryptophans (i.e., 117, 221 and 260 residues) with H. lanuginosa lipase are totally quenchable by acrylamide while completely inaccessible to iodide. On the contrary, the Trp89 residue is available for both quenchers. Using steady-state iodide fluorescence quenching data and the fluorescence-quenching-resolved-spectra (FQRS) method, the total emission spectrum of the native lipase has been decomposed into two spectral components. One of them, unquenchable by iodide, has a maximum of fluorescence emission at 330 nm and the second one, exposed to the solvent, emits at 338 nm. The resolved spectrum of the redder component corresponds to the Trp89 residue, which participates in about 65% of the total H. lanuginosa emission. The dynamic Stern—Volmer quenching constants calculated for both native (‘closed-lid’) and inhibited (‘open-lid’) lipase are 2.71 and 4.49 M −1 , respectively. The values obtained indicate that Trp89 is not deeply buried in the protein matrix. Our results suggest that distinct configurations of fungal lipase can be monitored using the fluorescence of the Trp89 residue located in the ‘lid’-helix which participates in an interfacial activation of the enzyme.

Steady-state and time-resolved fluorescence studies of fluorescent imprinted polymers

Abstract Molecular imprinting of synthetic polymers provides a powerful approach to control electronic and optical properties through nanoscale modification of molecular design of the material. The functional monomers were co-polymerised in the presence of a target/imprint molecule, which acts as a molecular template. Subsequent removal of the template molecules left behind functionalised cavities that are able to recognise the template molecule. The steady-state and time-resolved fluorescence spectroscopy studies are presented as methods for monitoring of the specificity and selectivity of binding of the template on the functionalised cavities.

Experimental and theoretical studies on solvent effects of amphiphilic conjugated polyenals

The spectroscopic properties of amphiphilic π-conjugated ω,ω′-(alkoxyphenyl)polyenals (alkoxy = methoxy, hexyloxy) containing from 1 to 8 double bonds have been studied both experimentally and theoretically. The studies considered the solvent effects on the spectral position and profiles of the absorption according to a great role of the solvent in photochemical processes. Positive solvatochromism in nonpolar solvents (2,2,4-trimethylpentane, CCl4, tetrahydrofuran, CH2Cl2) and negative solvatochromism in polar solvents (C2H5OH, CH3OH, CH3CN and dimethysulfoxide) suggest that an increase of solvent polarity causes a change of the canonical polyene structure—shift from a more polymethine-like to a more polyene-like state. Experimental findings were supported by theoretical calculations performed by AM1-CI/SM5.4/A method implemented in the AMSOL package from which dipole moments and free energy of solvation in ground μgs, ΔGgss and excited μex, ΔGexs state, respectively, proved that the continuum model of the solvent is satisfactory only for non-polar systems because in the polar solvents the specific solvent–solute interactions may introduce noticeably discrepancies in the solvation parameters. This theoretical method appeared useful to predict the critical parameter for the molecular admittance—HOMO–LUMO band gap—for conjugated polyenals in solvents of various polarity.

Photophysics of indole, tryptophan and N-acetyl-l-tryptophanamide (NATA): Heavy atom effect

Previously reported flash photolysis studies showed that the triplet state lifetime of aqueous indoles is μs long (12.5 μs for tryptophan [10]), while other recently reported phosphorescence lifetimes of aqueous indoles, determined from photon counting phosphorescence techniques, vary from μs (approximately 40 μs [11]) to ms (5 ms for indole [12]). This study was motivated to explain the discrepancy regarding the intrinsic triplet state lifetime of aqueous indole and its derivatives: tryptophan and N-acetyl-L-tryptophanamide (NATA). For this purpose, a new methodology based on both fluorescence and phosphorescence decay kinetics incorporating the heavy atom effect have been applied in order to determine some quantitative parameters of the photophysics of indole and its derivatives. Additionally, we have also determined the triplet state lifetimes of the studied indoles using flash photolysis in which contributions from both a first order component and a second order component (from triplet-triplet annihilation) have been taken into account in the triplet state depopulation. The measured phosphorescence lifetime of the indoles examined measures between the values reported by Fischer and Strambini and is consistent with the triplet state lifetime determined from flash photolysis. We hope that the results obtained in this paper would be helpful for deriving structural and dynamical information from phosphorescence data of tryptophan residues in proteins.

Temperature study of indole, tryptophan and N-acetyl-l-tryptophanamide (NATA) triplet-state quenching by iodide in aqueous solution

In this study, the temperature dependence of the measured phosphorescence lifetimes of aqueous indole, tryptophan and N-acetyl-L-tryptophanamide (NATA) between 6 and 55 °C in the absence and in the presence of iodide, a suitable intersystem crossing enhancer, has been determined. The obtained results suggest the existence of one process for the temperature-dependent, non-radiative deactivation of triplet states of the aqueous indoles in the absence of iodide. This process may be associated with the high sensitivity of indole triplet state lifetime to the subtle changes in the local viscosity of the surrounding aqueous environment or may be attributed to diffusional quenching by solvent molecules and/or by possible impurities present in water. However, the steep decrease in the measured phosphorescence lifetimes of indole and tryptophan with temperature suggests that diffusion-mediated quenching processes are not prevailing. Upon increasing concentration of iodide (up to 0.1 M), the obtained Arrhenius plots for the deactivation rate (1/τph) of the triplet states of the studied indoles were linear, which provided strong support for the hypothesis of the existence of one temperature dependent non-radiative process for the de-excitation of indoles triplet state. Our results showed that this process is attributed to the diffusion-controlled solute-quenching by iodide and, most probably, proceeds via reversibly formed exciplex. At concentration of iodide higher than 0.1M highly curved Arrhenius plots were obtained, which may indicate a change in the rate determining step with a change in temperature. This change most probably is associated with a transition from diffusion-controlled exciplex formation followed by rate-determining exciplex deactivation at high temperature.

Room temperature phosphorescence study on the structural flexibility of single tryptophan containing proteins

In this study, we have undertaken efforts to find correlation between phosphorescence lifetimes of single tryptophan containing proteins and some structural indicators of protein flexibility/rigidity, such as the degree of tryptophan burial or its exposure to solvent, protein secondary and tertiary structure of the region of localization of tryptophan as well as B factors for tryptophan residue and its immediate surroundings. Bearing in mind that, apart from effective local viscosity of the protein/solvent matrix, the other factor that concur in determining room temperature tryptophan phosphorescence (RTTP) lifetime in proteins is the extent of intramolecular quenching by His, Cys, Tyr and Trp side chains, the crystallographic structures derived from the Brookhaven Protein Data Bank were also analyzed concentrating on the presence of potentially quenching amino acid side chains in the close proximity of the indole chromophore. The obtained results indicated that, in most cases, the phosphorescence lifetimes of tryptophan containing proteins studied tend to correlate with the above mentioned structural indicators of protein rigidity/flexibility. This correlation is expected to provide guidelines for the future development of phosphorescence lifetime-based method for the prediction of structural flexibility of proteins, which is directly linked to their biological function.

Fluorescence studies of a silane gel to be applied as a carrier in optical sensors

Results of fluorescence studies on the degree of pyrene and aminopyrene elution from a silane gel by water and ethanol are discussed in the paper. The studies were carried out for gels obtained on glass plates that differed in composition. Silane monomers: TEOS—tetraethylorthosilane, APTES—3-aminopropyltriethoxysilane, GPTMS—3-glycidoxypropylmethoxysilane, PDMS—polydimethylsilane, TMAOH—tetraethylammonium hydroxide, water and ethanol were used. A fluorophore was trapped in the gel either physically or using covalent bonds with the amino group of a carrier and glutaraldehyde. Emission spectra of the probe steady-state fluorescence in the gel were recorded on subsequent days of storage of plates with the gel in fresh solvent. The kinetics of aminopyrene decay in the sol and silane gel was also recorded. On the basis of results obtained, the degree of elution of fluorophore from the gel was determined in view of the application of the silane gel as a carrier in the optical sensor and the nature of the probe environment.

Heavy atom induced phosphorescence study on the influence of internal structural factors on the photophysics of tryptophan in aqueous solutions.

In this study the effect of alanyl residue insertion into tryptophan and to some extent the effect of peptide bond on the photophysics of tryptophan chromophore has been studied. The photophysical parameters crucial in triplet state decay mechanism of aqueous AW, WA and AWA peptides have been determined applying our previously proposed methodology based on the heavy atom effect and compared with the previously reported values for tryptophan (Kowalska-Baron et al., 2012). The obtained results clearly indicated that the presence of alanyl residue and the peptide bond results in the changes in the fluorescence and phosphorescence decay kinetics of tryptophan. The fluorescence decays of the oligopeptides studied at pH 7 were biexponential. The longer lifetime component of WA arises from anionic form of this dipeptide, while the shorter one may be assigned to the zwitterionic form of WA. The observed invariance of the lifetimes of anionic and zwitterionic forms of WA throughout the pH studied supports the idea that these two components of WA fluorescence decay correspond to nearly independent species, possibly interconverting but at a rate slower than the fluorescence decay rates. Comparing the determined phosphorescence spectra of the oligopeptides studied with that of tryptophan, a slight blue-shift and more evident red-shift was observed in the spectrum of AW and WA, respectively. On the basis of the results of the phosphorescence measurements performed at pH 10, the 170 μs lifetime of WA, observed even at pH 7, may be assigned to the anionic form of the compound. It may be suggested that at pH 7 during the excited triplet state lifetime of WA there is a shift in the equilibrium towards the anionic form of this dipeptide. In the case of AW and AWA at pH 7 the obtained monoexponential decay kinetics, most probably, arise from zwitterionic forms of these peptides. The determined triplet quantum yield of AWA is slightly lower than that of tryptophan, while the quantum yield of AW is twofold lower than that of tryptophan. The highest value of the determined triplet quantum yield of WA confirms the presence of anionic form of this dipeptide at pH 7.

Photophysics of indole-2-carboxylic acid (I2C) and indole-5-carboxylic acid (I5C): heavy atom effect.

In this study the effect of carboxylic group substitution in the 2 and 5 position of indole ring on the photophysics of the parent indole chromophore has been studied. The photophysical parameters crucial in triplet state decay mechanism of aqueous indole-2-carboxylic acid (I2C) and indole-5-carboxylic acid (I5C) have been determined applying our previously proposed methodology based on the heavy atom effect and fluorescence and phosphorescence decay kinetics [Kowalska-Baron et al., 2012]. The determined time-resolved phosphorescence spectra of I2C and I5C are red-shifted as compared to that of the parent indole. This red-shift was especially evident in the case of I2C and may indicate the possibility of hydrogen bonded complex formation incorporating carbonyl CO, the NH group of I2C and, possibly, surrounding water molecules. The possibility of the excited state charge transfer process and the subsequent electronic charge redistribution in such a hydrogen bonded complex may also be postulated. The resulting stabilization of the I2C triplet state is manifested by its relatively long phosphorescence lifetime in aqueous solution (912 μs). The relatively short phosphorescence lifetime of I5C (56 μs) may be the consequence of more effective ground-state quenching of I5 C triplet state. This hypothesis may be strengthened by the significantly larger value of the determined rate constant of I5C triplet state quenching by its ground-state (4.4 × 10(8)M(-1)s(-1)) as compared to that for indole (6.8 × 10(7)M(-1)s(-1)) and I2C (2.3 × 10(7)M(-1)s(-1)). The determined bimolecular rate constant for triplet state quenching by iodide [Formula: see text] is equal to 1 × 10(4)M(-1)s(-1); 6 × 10(3)M(-1)s(-1) and 2.7 × 10(4)M(-1)s(-1) for indole, I2 C and I5 C, respectively. In order to obtain a better insight into iodide quenching of I2C and I5C triplet states in aqueous solution, the temperature dependence of the bimolecular rate constants for iodide quenching of the triplet states has been expressed in Arrhenius form. The linearity of the obtained Arrhenius plots clearly indicated the existence of one temperature-dependent non-radiative process for the de-excitation of I2C and I5C triplet state in the presence of iodide. This process may be attributed to the solute-quenching by iodide and, most probably, proceeds via reversibly formed exciplex. The activation energies obtained from linear Arrhenius plots (1.89 kcal/mol for I5 C; 2.55 kcal/mol for I2 C) are smaller as compared to that for diffusion controlled reactions in aqueous solution (about 4 kcal/mol), which may indicate the great importance of the electrostatic interactions between solute and iodide ions in lowering the energy barrier needed for the formation of the triplet-quencher complex. Based on the theoretical predictions (at the DFT(CAM-B3LYP)/6-31+G(d,p) level of theory) and careful analysis of the obtained FTIR spectra it may be concluded that in the solid state I2 C and I5 C molecules form associates by intermolecular NH · · · OC and OH · · · OC hydrogen bonding interactions, whereas the existence of intramolecular NH · · · OC interactions in the solid state of I2C and I5C is highly unlikely.