Non-amphiphilic Derivatives Research Articles

Self-organization of non-amphiphilic molecules. Studies of thin films of long-chain homologous dialkylthioethers at the water/air interface

In contrast to classical surfactants, the knowledge about the self-organization of alkanes and their hydrophobic derivatives is still limited. In this paper, we present the results of the studies of self-assembly of long-chain dialkylthioethers at the air/water interface. The substitution of one methylene group by the thioether divalent sulfur introduces significant dipole moment to the alkane chain without affecting the hydrophobicity, which profoundly influences the self-assembly of these molecules. Depending on the location of the thioether group in the hydrophobic chain, the investigated molecules can form Langmuir monolayers, which are stabilized by the thioether-water H-bonds formation, or random multilayers. The structures of the monolayers were investigated with the application of Grazing Incidence X-ray Diffraction. To elucidate important structural differences between thioether and alkane monolalyers of the same hydrocarbon chain length, we applied the methods of quantum chemistry (ETS–NOCV calculations). It turned out that the introduction of one sulfur atom affects the distribution of electron density not only in the proximity of this atom but generally along the chain. The combination of experimental and calculation methods provides to the better understanding of the fundamental question of the self-organization of long-chain alkanes and their non-amphiphilic derivatives at interfaces.

Photophysical properties of 4-methyl 3-phenyl coumarin organized in Langmuir–Blodgett films: Formation of aggregates

Langmuir–Blodgett (LB) films of 4-methyl 3-phenyl coumarin (MPC), a non-amphiphilic derivative of coumarin mixed with stearic acid (SA) are studied. The surface pressure–area ( π– A) isotherm study at air–water interface reveals a repulsive type of interaction between the MPC and SA molecules resulting in the formation of aggregates of MPC molecules. Absorption, fluorescence and phosphorescence studies indicate the formation of J-type aggregate in LB film. Low temperature (77 K) phosphorescence lifetime is found to be decreased due to aggregate formation. Micro structured aggregates are observed after examining the surface morphology of LB film by scanning electron microscopy (SEM).

Interaction of Pyrene-1-Carboxaldehyde with micelles and mixed micelles of polyoxyethylene nonyl phenol (Igepal): A spectroscopic study

The absorption and steady-state fluorescence of a non-amphiphilic derivative of pyrene, Pyrene-1-Carboxaldehyde (1-PyCHO) is sensitive to the probe's environment. Association of the surfactants in aqueous medium as micelles and mixed micelles has been studied from the photophysical changes of 1-PyCHO in these media. The physicochemical characteristics of the micelles, i.e. CMC, aggregation number have been determined. The obtained CMC values from different methods are close to each other. The association constants of the 1-PyCHO molecules with the non-ionic micelle of Igepal and location of the fluorophore in the micellar environment have been determined. Micropolarity of Igepal micelle and Igepal/Igepal mixed micelle surrounding 1-PyCHO has been ascertained comparing the shift in fluorescence maximum with E T(30) values of standard solutions.

Spectroscopic characterizations of non-amphiphilic 2-(4-biphenylyl)-6-phenyl benzoxazole molecules at the air–water interface and in Langmuir–Blodgett films

This communication reports about the successful incorporation of a well-known non-amphiphilic derivative of oxazole chromophore 2-(4-biphenylyl)-6-phenyl benzoxazole abbreviated as PBBO, in Langmuir–Blodgett films when mixed with stearic acid (SA) as well as also an inert polymer matrix polymethylmethacrylate (PMMA). The surface pressure versus area per molecule isotherms of the Langmuir films of PBBO mixed with PMMA or SA at different mole fractions reveal that the area per molecule decreases consistently with increasing mole fractions of PBBO. Area per molecule versus mole fraction curve shows that the experimental data points coincide with the ideality curve predicted by the additivity rule, which leads to the conclusion of either ideal mixing or complete demixing of the binary components. The UV-vis absorption and fluorescence spectroscopic studies of mixed LB films of PBBO reveal the nature of complete demixing of the binary components of the sample molecules (PBBO) and PMMA or SA molecules. This complete demixing leads to the formation of clusters and aggregates of PBBO molecules in Langmuir and Langmuir–Blodgett films. J-type aggregates of PBBO molecules in LB films have been confirmed by UV-vis absorption spectroscopic study. Aggregation of PBBO molecules in LB films giving rise to excimeric emission has been demonstrated by fluorescence spectroscopic study. Excitation spectroscopic study clearly confirmed the presence of excimeric sites.

Spectroscopic properties of non-amphiphilic 1-pyrene carboxaldehyde assembled in Langmuir–Blodgett films

1-Pyrene carboxaldehyde (abbreviated as PyCHO), a non-amphiphilic derivative of pyrene has been successfully incorporated in Langmuir–Blodgett (LB) films, mixed with stearic acid (SA). Surface pressure ( π) vs. area per molecule isotherms of PyCHO mixed with SA at different mole fractions reveal a plateau like region which indicates a phase transition corresponding to a reorientation of the PyCHO molecules at the air–water interface. Moreover, a positive deviation from the ideal behaviour of the area per molecule in different mole fraction in the mixed film at the air–water interface clearly reveals a repulsive interaction between the components resulting in the formation of aggregates of PyCHO molecules in the mixed films. Unlike pyrene molecules, formation of J-aggregates of PyCHO molecules in the mixed LB films has been confirmed by absorption spectroscopic studies. The fluorescence spectrum of the LB mixed film of PyCHO has been observed to lie between the solution and microcrystalline spectra and has a combination of both monomeric and excimeric species. A striking similarity between the LB film emission spectra and the emission spectra of PyCHO in ethanol–water binary mixture confirms the formation of low dimensional microcrystalline aggregates in the LB film.

Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris.

In the methylotrophic yeast Pichia pastoris, we expressed the rat acetylcholinesterase H and T subunits (AChEH and AChET respectively), as well as truncated subunits from rat (W553stop or AChETDelta, from which most of the T-peptide was removed) and from Bungarus (V536stop, or AChENAT, or AChEDelta, reduced to the catalytic domain). We show that AChEH and AChET subunits are processed into the same molecular forms as in vivo or in transfected mammalian cells, but that lytic processes converting amphiphilic forms into non-amphiphilic derivatives appear to be more active in yeast. The production of glycophosphatidylinositol (GPI)-anchored molecules (dimers, with a small proportion of monomers) demonstrates that P. pastoris can correctly process a mammalian C-terminal GPI-addition signal. Truncated rat and Bungarus AChE molecules, which exclusively generated non-amphiphilic monomers, were released more efficiently and thus produced more AChE activity. In the hope of increasing the production of AChE, we replaced the endogenous signal peptide by yeast prepeptides, with or without a propeptide. We found that the presence of a propeptide, which does not exist in AChE, does not prevent the proper folding of the enzyme, and that it may either increase or decrease the yield of secreted AChE, depending on the signal peptide. Surprisingly, the highest yield was obtained with the endogenous signal peptide. For all combinations, the yield was 2-3 times higher for Bungarus than for rat AChE, probably reflecting differences in the folding efficiency or stability of the polypeptides. The Michaelis constant (Km), the constant of inhibition by excess substrate (Kss) and the catalytic constant (kcat) values of the recombinant AChEs obtained both in P. pastoris and in COS cells, were essentially identical with those of the corresponding natural enzymes, and the Ki values of active-site and peripheral-site inhibitors (edrophonium, decamethonium, propidium) were similar.

Subcellular distribution of acetylcholinesterase forms in chromaffin cells

The presence of acetylcholinesterase (AChE) in chromaffin granules has been controversial for a long time. We therefore undertook a study of AChE molecular forms in chromaffin cells and of their distribution during subcellular fractionation. We characterized four main AChE forms, three amphiphilic forms (Ga1, Ga2 and Ga4), and one non-amphiphilic form (Gna4). Each form shows the same molecular characteristics (sedimentation, electrophoretic migration, lectin interactions) in the different subcellular fractions. All forms are glycosylated and seem to possess both N-linked and O-linked carbohydrate chains. There are differences in the structure of the glycans carried by the different forms, as indicated by their interaction with some lectins. Glycophosphatidylinositol-specific phospholipases C converted the Ga2 form, but not the other amphiphilic forms, into non-amphiphilic derivatives. The distinct patterns of AChE molecular forms observed in various subcellular compartments indicate the existence of an active sorting process. Gna4 was concentrated in fractions of high density, containing chromaffin granules. We obtained evidence for the existence of a lighter fraction also containing chromogranin A, tetrabenazine-binding sites and Gna4 AChE, which may correspond to immature, incompletely loaded granules or to partially emptied granules. The distribution of Gna4 during subcellular fractionation suggested that this form is largely, but not exclusively, contained in chromaffin granules, the membranes of which may contain low levels of the three amphiphilic forms.

Absence of substrate inhibition and freezing-inactivation of the mosquito acetylcholinesterase are caused by alterations of hydrophobic interactions

Membrane-bound acetylcholinesterase (AChE) from mosquito showed the characteristic substrate inhibition of this enzyme, but 105000×g supernatants of freshly extracted enzyme did not. Addition of chaotropic anions, a freeze-thaw cycle and autolysis of the amphiphilic acetylcholinesterase to its non-amphiphilic derivatives resulted in return of the substrate inhibition feature along with an apparent increment in the enzyme activity. These results suggested that the lipidic environment of the mosquito AChE is temporarily perturbed when extracted. The enzyme is probably trapped in non-sedimenting mixtures composed of endogenous amphiphilic molecules. The occurrence of this phenomenon was not affected by the presence of Triton X-100 and other detergents, either alone or in combination with sodium chloride. Freezing in the presence of strong chaotropic anions (perchlorate, iodide and thiocyanate) caused the irreversible inactivation of the mosquito AChE. Crude and incomplete purified fractions of the enzyme were more sensitive than a more purified preparation. With both the purified AChE and the non-purified AChE, amphiphilic AChE was more freeze labile. Freezing at −10°C enhanced inactivation of non-purified fractions. At this temperature, even weak chaotropic anions (fluoride, chloride and nitrate), while in combination with non-ionic detergents that solubilized mosquito AChE efficiently, reduced the enzyme activity of these fractions. In this case, recovery of the enzyme activity by incubation at 25°C was inversely correlated with the effectiveness of the chaotropic anion. Gel filtration failed to show any change in the hydrodynamic radius of the freezing-inactivated AChE. Therefore, this phenomenon is explained as different degrees of denaturation of the enzyme in direct association with the chaotropic strength. Thus, antichaotropic anions, such as sulfate, should improve the stability of the mosquito acetylcholinesterase during extraction, purification and storage.

Amphiphilic and nonamphiphilic forms of Torpedo cholinesterases: I. Solubility and aggregation properties.

We report an analysis of the solubility and hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. We distinguish globular nonamphiphilic forms (Gna) from globular amphiphilic forms (Ga). The Ga forms bind micelles of detergent, as indicated by the following properties. They are converted by mild proteolysis into nonamphiphilic derivatives. Their Stokes radius in the presence of Triton X-100 is approximately 2 nm greater than that of their lytic derivatives. The G2a forms fall in two classes. Class I contains molecules that aggregate in the absence of detergent, when mixed with an AChE-depleted Triton X-100 extract from electric organ. AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which is also detectable in detergent-soluble (DS) extracts of electric lobes and spinal cord. Class II forms never aggregate but only present a slight shift in sedimentation coefficient, in the presence or absence of detergent. This class contains the AChE G2a forms of plasma and of the low-salt-soluble (LSS) fractions from spinal cord and electric lobes. The heart possesses a BuChE G2a form of class II in LSS extracts, as well as a similar G1a form. G4a forms of AChE, which are solubilized only in the presence of detergent and aggregate in the absence of detergent, represent a large proportion of cholinesterase in DS extracts of nerves and spinal cord, together with a smaller component of G4a BuChE. These forms may be converted to nonamphiphilic derivatives by Pronase. Nonaggregating G4a forms exist at low levels in the plasma (BuChE) and in LSS extracts of nerves (BuChE) and spinal cord (AChE).

Amphiphilic and nonamphiphilic forms of Torpedo cholinesterases: II. Electrophoretic variants and phosphatidylinositol phospholipase C-sensitive and -insensitive forms.

We report an electrophoretic analysis of the hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. In charge-shift electrophoresis, the rate of electrophoretic migration of globular amphiphilic forms (Ga) is increased at least twofold when the anionic detergent deoxycholate is added to Triton X-100, whereas that of globular nonamphiphilic forms (Gna) is not modified. The G2a forms of the first class, as defined by their aggregation properties, are converted to nonamphiphilic derivatives by phosphatidylinositol phospholipase C (PI-PLC) and human serum phospholipase D (PLD). AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which also exists in very small quantities in detergent-solubilized extracts of electric lobes and spinal cord. They present different electrophoretic mobilities, so that each of these tissues contains a distinct "electromorph," or two in the case of electric organs. The G2a forms of the second class (AChE in plasma, BuChE in heart), as well as G4a forms of AChE and BuChE, are insensitive to PI-PLC and PLD but may be converted to nonamphiphilic derivatives by Pronase.