Horse heart cytochrome c entrapped into the hydrated liquid-crystalline phases of phytantriol: X-ray diffraction and Raman spectroscopic characterization.

Small angle X-ray diffraction (SAXD), resonance Raman (RR) spectroscopy with 413 nm excitation, and non-resonance Raman technique with 785 nm excitation were used to probe the influence of entrapped cytochrome c (Cyt c) on the structure of hydrated phytantriol (Phyt) liquid-crystalline phases as well as conformational changes of heme group and secondary structure of the protein. SAXD measurements indicated that incorporation of Cyt c affects both nanostructure dimensions and type of liquid-crystalline phases of hydrated Phyt. The unit cell dimensions decrease with increasing Cyt c concentration for all phases. In addition, protein perturbs the nanostructure of Q(230) and Q(224) liquid-crystalline phases of hydrated Phyt to such an extent that they transform into the Q(229) phase with the Im3m space group. RR data revealed that entrapment of oxidized Cyt c into the Q(230) phase at 1 wt.% content results in near complete reduction of central iron ion of the heme group, while its low-spin state and six-ligand coordination configuration are preserved. Based on the analysis of heme out-of-plane folding vibration near 568 cm(-1) (γ(21)) and ν(48) mode at 633 cm(-1), it was demonstrated that the protein matrix tension on the heme group is relaxed upon incorporation of protein into Q(230) phase. Non-resonant Raman bands of difference spectra showed the preservation of α-helix secondary structure of Cyt c in the liquid-crystalline phase at relatively high (5 wt.%) content. The Cyt c induced spectroscopic changes of Phyt bands were found to be similar as decrease in temperature.

Enzymatic activity of lipase-nanoparticle conjugates and the digestion of lipid liquid crystalline assemblies.

Variants of lipase were attached to gold nanoparticles (NPs) and their enzymatic activity was studied. The two bioengineered lipase variants have been prepared with biotin groups attached to different residues on the protein outer surface. The biotinylation was evidenced by denaturing polyacrylamide gel electrophoresis and quantified by the ([2-(4'-hydroxyazobenzene)]benzoic acid spectrophotometric test. NPs of 14 +/- 1 nm diameter coated with thiolated-polyethylene glycol ligands containing controlled proportions of biotin moieties have been prepared and characterized by transmission electron microscopy, UV-vis spectroscopy, small angle neutron scattering, and elemental analysis. These biotin-functionalized NPs were conjugated to lipase using streptavidin as a linker molecule. Enzyme activity assays on the lipase-nanoparticle conjugates show that the lipase loading and activity of the NPs can be controlled by varying the percentage of biotin groups in the particle protecting coat. The lipase-NP conjugates prepared using one variant display higher activity than those prepared using the other variant, demonstrating orientation-dependent enzyme activity. Cryogenic transmission electron microscopy was used to visualize the enzymatic activity of lipase-NP on well-defined lipid substrates. It was found that lipase-coated NPs are able to digest the substrates in a different manner in comparison to the free lipase.

Lipophilic 1,4-naphthoquinone derivatives: synthesis and redox properties in solution and entrapped in the aqueous cubic liquid-crystalline phase of monoolein.

1,4-naphthoquinone derivatives (NQD) containing lipophilic alkyl chains, i.e. 2-((Z)-heptadec-8-enyl)-3-methyl 1,4-naphthoquinone (QMe), 2-((Z)-heptadec-8-enyl)-3-hydroxy-1,4-naphthoquinone (QOH) and (Z)-octadec-9-enyl 1,4-naphthoquinone-2-carboxylate (QE) were synthesized. The redox behavior of these NQD was studied in ethanol and entrapped in the reversed bicontinuous cubic phase of space group type Pn3m (Q(224)) of aqueous monoolein. In ethanol, cyclic voltammetry curves exhibit two pairs of oxidation-reduction peaks arising from the redox processes controlled by adsorption and molecular diffusion. The NQD molecules are also electrochemically active in the cubic phase, indicating the participation in the 2e(-), 2H(+)-redox cycle at pH<9. Therefore, it was concluded that the head group of the entrapped NQD reaches the lipid bilayer interface of cubic phase during the process.

Anti-inflammatory activity of some potassium salts of N,N-disubstituted 4-aminoazobenzenesulfonic acids in rat adjuvant arthritis.

Anti-inflammatory effects of three potassium salts of N,N-disubstituted 4-aminoazobenzenesulfonic acids were investigated and compared to that of acetylsalicylic acid (ASA) in rats with adjuvant arthritis (AA). Prophylactic oral administration of all compounds in a dose of 150 mg/kg ameliorated AA symptoms in animals. The most pronounced anti-inflammatory activity at the end of the experiment showed compound 1 containing imino group: it significantly suppressed joint swelling by 48.2% in female and by 44.2% in male rats with AA. The development of polyarthritis after the treatment with this compound was the lowest in female (20%) and male (40%) rats (in control--100% of animals with polyarthritis). All derivatives, and especially compound 1, also improved the systemic parameters of disease such as blood indices and internal organs' weight, and showed no toxicity on the main organs such as liver and spleen.

Probing of lipase activity at air/water interface by sum-frequency generation spectroscopy.

The infrared-visible sum-frequency generation (SFG) vibrational spectroscopy was used to probe enzymatic activity of Thermomyces lanuginosus lipase (TLL) at air/water interface. A monolayer of amphiphilic O-palmitoyl-2,3-dicyanohydroquinone (PDCHQ), containing target ester group and two CN groups serving as vibrational markers, was utilized as an enzyme substrate. SFG data revealed the detailed molecular scale structure and properties of the PDCHQ layer at the interface. In particular, we demonstrate that hydrophilic headgroup of PDCHQ is mainly in the form of an oxyanion, and the enzyme-induced cleavage of the ester bond could be spectroscopically monitored by the disappearance of the intense C tripple bond N resonance at 2224 cm(-1). The enzymatic nature of the ester bond cleavage was confirmed by the control experiments with deactivated S146A mutant variant of TLL. By comparing action of wild type (WT) TLL and its inactive S146A mutant, it was shown that two effects take place at the interface: disordering of the lipid monolayer due to the adsorption of enzyme and enzymatic cleavage of the ester bond. The concentration of enzyme as low as 10 nM could be easily sensed by the SFG spectroscopy. We present spectroscopic evidence that upon hydrolysis one of the products, 2,3-dicyanohydroquinone, leaves the surface, while the other, palmitic acid, remains at air/water interface in predominantly undissociated form with the mono-hydrogen-bonded carbonyl group. Strong amide I (1662 cm(-1)) and amide A (3320 cm(-1)) SFG signals from TLL suggest that enzyme molecules position themselves at air/water interface in an orderly fashion. Presented work demonstrates the potential of SFG spectroscopy for in situ real-time monitoring of enzymatic processes at air/water interface.

Thermomyces lanuginosus lipase in the liquid-crystalline phases of aqueous phytantriol: X-ray diffraction and vibrational spectroscopic studies.

The influence of Thermomyces lanuginosus lipase (TLL) on the phase behaviour of liquid-crystalline phases of aqueous phytantriol as well as conformational changes of TLL entrapped in the cubic Q230 phase have been studied by small angle X-ray diffraction (SAXD), FT-Raman, and FT-IR techniques. It was found that the lipidic Q230 phase is able to accommodate up to 10 wt.% of TLL, and the temperature of phase transition to the inverted hexagonal phase H(II) increases indicating stabilizing effect of the protein. FT-Raman analysis of Trp amino acid marker band W3 revealed that the average rotation angle around the C3-Cbeta bond of four Trp residues of TLL in the Q230 phase increases. Reasoning from available TLL crystallographic data, this result is explained by structural transition of entrapped protein to so-called "open" and more related to the enzymatically-active conformation. TLL secondary structure analysis by amide I and amide III vibrational bands showed that content of alpha-helixes does not change, while a part of beta-sheet structures transforms to less ordered elements upon incorporation of protein into the Q230 phase of aqueous phytantriol.

Computational Modeling of the Electrochemical System of Lipase Activity Detection.

This paper presents computational modeling of response kinetics of bioelectroanalytical system based on solid supported lipase substrate and lipase interaction. The model assumes that lipase substrate is formed by dripping and drying a small amount of the ethanol solution of 9-(5'-ferrocenylpentanoyloxy)nonyl disulfide (FPONDS) and that lipase is capable of cleaving FPONDS ester bonds via hydrolysis mechanism. Two mathematical models have been developed and evaluated trough computational simulation series by comparing them to experimental data. The results of simulation demonstrate that a good fitting might be obtained only taking into account non-linear substrate wash off process.

Mediated oxidation of ascorbic acid on a homologous series of ferrocene-terminated self-assembled monolayers.

The kinetics of electrocatalytic oxidation of ascorbate was studied on a series of redox self-assembled monolayers (SAMs) of the general formula Fc(CH2)4COO(CH2)nSH as electron-transfer mediators, where Fc is the ferrocenyl group and n = 3, 6, 9, and 11. We show that the rate of electron transfer from ascorbate to the surface-confined Fc+ decreases with increasing n. The rationale for the dependence of the rate of electrocatalytic activity and n, in the presence of ClO4, is obtained from Fourier-transform surface-enhanced Raman spectroscopy (FT-SERS), cyclic voltammetry, and electrochemical quartz crystal microbalance (EQCM) data. In particular, FT-SERS shows decreasing amounts of surface-bound ClO4- upon oxidation of the ferrocene with decreasing n, while EQCM data show the effective electrode mass increase was consistently higher on the shorter chain SAMs. This mass increase is likely due to increasing ferricinium cation hydration. As n decreases, the SAMs become less ordered (FT-SERS data), as is widely known from previous literature. Disorder favors water penetration into the SAM, which, in turn, increases the hydration of the Fc+ (EQCM data). Increased hydration of the Fc+ impedes the formation of Fc+-ClO4- ion pairs (EQCM and FT-SERS data), which, consequently, accelerates the electrocatalytic electron transfer from the solution-dissolved ascorbate.

Bionanoconjugation via click chemistry: The creation of functional hybrids of lipases and gold nanoparticles.

A simple and versatile method for the preparation of functional enzyme-gold nanoparticle conjugates using "click" chemistry has been developed. In a copper-catalyzed 1,2,3-triazole cycloaddition, an acetylene-functionalized Thermomyces lanuginosus lipase has been attached to azide-functionalized water-soluble gold nanoparticles under retention of enzymatic activity. The products have been characterized by gel electrophoresis and a fluorometric lipase activity assay. It is estimated that the equivalent of approximately seven fully active lipase molecules are attached to each nanoparticle.

Infrared and Raman bands of phytantriol as markers of hydrogen bonding and interchain interaction.

Hydrogen bonding and interchain interactions in phytantriol, 3,7,11,15-tetramethyl-1,2,3-hexadecanetriol, have been studied by Fourier transform infrared (FT-IR) and Raman spectroscopies. Assignments of the bands were performed based on the OH/OD isotopic substitution, molecular modeling, and measurements of polarized Raman spectra. Marker bands were evaluated from the temperature-dependent spectral changes. It is shown that Raman spectroscopy provides sensitive markers, namely I(delta(CH2))/I(deltas(CH3)), tau(CH)2, I(nus(CH3)(FR))/I(nus(CH2)), and nus(CH2) for probing the interactions between the hydrocarbon chains. Hydrogen bonding interaction might be studied through the difference Raman spectroscopy by the analysis of polarized band at 811 cm-1. Relationship is found between the frequencies of IR bands at 883-873 and 1097-1086 cm-1, associated with the vibrations localized at the primary COH site, and the frequencies of OH stretching mode, making these bands specific markers in the analysis of hydrogen bonding. Evaluated marker bands may be of utility to probe the interchain and hydrogen bonding interaction of phytantriol with guest molecules in the practically important aqueous liquid-crystalline phases of this lipid.

Electrochemical method for the detection of lipase activity.

A novel electrochemical technique for the general assay of lipase activity is described. The method utilizes a solid-supported lipase substrate, which is formed by dripping and drying a small amount of an ethanol solution of 9-(5'-ferrocenylpentanoyloxy)nonyl disulfide (FPONDS) onto gold modified by a hexanethiol self-assembled monolayer. The redox ferrocene group of FPONDS generates the electrochemical signal, the intensity of which is proportional to the number of FPONDS molecules at the interface. Electrochemical and surface-enhanced infrared absorption spectroscopic data, as well as control experiments with an engineered, deactivated mutant enzyme, demonstrate that the wild-type lipase from Thermomyces lanuginosus is capable of cleaving the ester bonds of FPONDS molecules via an enzymatic hydrolysis mechanism, which includes the adsorption of the lipase onto the substrate surface. The hydrolysis liberates the ferrocene groups from the interface triggering a decay of the electrochemical redox signal. The rate of the electrochemical signal decrease is proportional to the lipase activity/concentration. These data suggest a general method for the direct measure of enzymatic activity of lipases.

Interactions of Cyclic AMP and Its Dibutyryl Analogue with a Lipid Layer in the Aqueous Mixtures of Monoolein Preparation and Dioleoyl Phosphatidylcholine as Probed by X-Ray Diffraction and Raman Spectroscopy.

Interactions of adenosine 3':5'-cyclicmonophosphate (cAMP) andN(6),2'-O-dibutyryladenosine3':5'-cyclic monophosphate (dbcAMP) with alipid layer composed of monoolein-basedpreparation and dioleoylphosphatidylcholine (DOPC) wereinvestigated by small-angle X-raydiffraction (SAXD) and Raman spectroscopy.The reversed hexagonal (H(II))MO/DOPC/H(2)O phase of 65:15:20 wt.%composition was selected as a referencesystem. SAXD revealed that entrapment (atthe expense of water) of 3 wt.% cAMP intothe reference system did not change thepolymorphic form and structural parametersof the phase. The same content of dbcAMPinduced the transition from the H(II)phase to the reversed bicontinuous cubicphase of space group Ia3d. Thistransition is explained by the increase oflipid head-group area due to thepenetration of the acylated adenine groupof dbcAMP into the polar/apolar region oflipid layer. The conclusion is supported byRaman spectroscopy, showing thedisruption/weakening of hydrogen bonding inthe MO/DOPC-based matrix at the N1- andN3-sites of the dbcAMP adenine ring. Asdistinct from dbcAMP, cAMP remains mostlyin the water channels of the H(II)phase, although the phosphate residue ofnucleotide interacts with the quaternaryammonium group of DOPC. Both nucleotidesincrease the population of gaucheisomers in the DOPC choline group.

Synthesis and fungicidal activity of lipophylic N- and O-acyl derivatives of beta-hydroxy DL-alpha-amino acids.

Synthesis of N- and O-acyl derivatives of DL-serine and threo-DL-phenylserine was accomplished by a regioselective acylation of the corresponding amino acid. The residues introduced into amino acid structure contain hydrophobic long chain or aromatic, namely lauroyl, myristoyl and phenylacetyl moieties. The fungicidal activity against six strains of fungi was studied. Several compounds were found to be effective against growth of fungi, and O-myristoyl-DL-serine 2 and N-phenylacetyl-threo-DL-phenylserine 8 completely inhibited the growth of the mycelium of the fungus Verticillium dahliae.

Towards redox active liquid crystalline phases of lipids: a monoolein/water system with entrapped derivatives of ferrocene.

The phase and electrochemical behavior of the aqueous mixtures of monoolein (MO) and synthetic ferrocene (Fc) derivatives containing long alkyl chains-(Z)-octadec-9-enoylferrocene (1), (Z)-octadecen-9-ylferrocene (2), and ferrocenylmethyl (Z)-octadec-9-enoate (3)-were studied. At low hydration, the reversed micelles (L(2) phase) and cubic Q(230) phase of MO can accommodate relatively high amounts (>6 wt.%) of the Fc-derivative 2, whereas at high hydration, the pseudoternary cubic phase Q(224) is destabilized even at about 2 wt.% of this Fc. Increasing the Fc-derivative content induces L(alpha)-->L(2) and L(alpha)-->reversed bicontinuous cubic phase (Q(II))-->H(II) transitions depending upon hydration. A rough study of the MO system containing compounds 1 and 3 indicates very similar phase behavior to that of the MO/2/H(2)O system. Compound 2 apparently has no effect on the lipid monolayer thickness in the pseudoternary L(alpha), H(II) and Q(II) liquid crystalline phases of MO. Within a 3D-structure of the Q(224) phase, derivatives 1-3 exhibit electrochemical activity on the gold electrode. The one-electron redox conversion processes are electrochemically quasi-reversible and controlled by diffusion. The values of apparent diffusion coefficient (D(app)) and heterogeneous electron-transfer rate constant (k(s)) of Fcs are significantly lower in the cubic phase matrix when compared to the acetonitrile solution. By contrast, the MO H(II) phase with entrapped Fc-derivatives does not exhibit electrochemical activity on the electrode surface. It is suggested that the diffusional anisotropy and/or localized aggregation of compounds 1-3 within a 2D-structure of the H(II) phase account(s) for the latter observation.