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In the last few decades, the preparation of solid-supported lipid bilayers by immersing a solid substrate in an aqueous solution where the lipid is dissolved with the aid of a surfactant, followed by dilution of the solution, has been reported. In this study, we attempted to interpret the evolution of supported surfactant/lipid assemblies towards the supported lipid bilayer in terms of a phase equilibrium between the supported assembly phase and its ambient solution system consisting of the dispersed surfactant/lipid assembly phase and the bulk solution phase comprising monomeric surfactant and lipid. We characterized the supported assembly formed on hydrophilized Ge or mica substrates in equilibrium with aqueous solutions containing various concentrations of the nonionic surfactant, n-octyl-ß-D-glucopyranoside (OG) and the amphoteric phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), using interaction-force-profile measurements by atomic force microscopy (AFM), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). We also investigated the ambient solution system using equilibrium dialysis to obtain the partition equilibrium profile of OG between the bulk solution and dispersed assembly phases in the micellar or vesicular states. These studies indicate that the properties of the supported assembly depend on the composition of the dispersed assembly and concentration of monomerically dissolved OG. Further, a type of micellar-bilayer state transition occurs in the supported assembly, roughly synchronized with that in the dispersed assembly.

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In the preceding paper, we investigated a mixed assembly composed of a nonionic surfactant, n-octyl-ß-D-glucopyranoside (OG), and an amphoteric lipid, 1,2-dioleolyl-sn-glycero-3-phosphocholine (DOPC), formed on hydrophilized solid substrates immersed in aqueous solutions containing OG and DOPC. The experimental data could be interpreted in terms of the phase equilibrium; thus, the partition equilibrium profile of OG between the bulk solution phase and the supported assembly phase was obtained, as well as that between the bulk solution and the dispersed assembly. The partition equilibrium profiles suggested that micellar-bilayer state transitions occur both in the supported assembly and in the dispersed one in a roughly synchronized manner, even though there are significant discrepancies between them. In this paper, we propose a simple thermodynamic model for the micellar-bilayer transition of the dispersed and supported assembly of OG and DOPC, assuming that the micellar and bilayer states are also pseudo-phases distinct from each other. Using this model, we analyzed these partition equilibrium profiles and concluded that the transition in the supported assembly should mainly be attributed to the transition in the dispersed assembly, which is partly modified by the interaction energy between the supported assembly and the substrate.

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A disulfide-deficient variant of hen lysozyme, 0SS, is known to form an amyloid protofibril spontaneously, and to dissociate into monomers at high hydrostatic pressure. We carried out native PAGE at various temperatures (20-35°C) and pressures (0.1-200 MPa), to characterize the dissociation equilibrium of disulfide-deficient variant of hen lysozyme amyloid protofibril. Based on the density profiles, the partial molar volume and thermal expansibility changes for dissociation, ΔvD and ΔeD , were obtained to be -74 cm(3) /mol at 25°C and -2.3 cm(3) mol(-1) K(-1) , respectively. The dissociation of amyloid fibril destroys the cross ß-structure, and such conformational destruction in native protein fold rarely accompanies negative thermal expansibility change. We discussed the negative thermal expansibility change in terms of hydration and structural packing of the amyloid protofibril.

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It has been demonstrated that differential conductivity, dκ/dC, is useful for experimentally extracting the contribution of micellar aggregates from the conductivity data of an ionic surfactant solution in which aggregates and monomers coexist. This extraction allows us to treat the micellization process using a simple two-state model (nS→M) instead of the general mass action model of micellization (nS+qG→M). As a result, the three parameters of micellization, i.e., aggregation number (n), micellization constant, and ionization degree (α) of micelles, for homologous double-chain surfactants and bile salt derivatives can be determined. It was found that when the side-chain was long enough, the double-chain surfactants examined formed highly ionized (α=0.6-0.9) and small (ca. n=20) aggregates, regarded as premicelles.

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Two new applications of the recently developed technique of composition gradient static light scattering (CG-SLS) are presented. 1), The method is demonstrated to be capable of detecting and quantitatively characterizing reversible association of chymotrypsin and bovine pancreatic trypsin inhibitor in a solution mixture and simultaneously occurring reversible self-association of chymotrypsin at low pH in the same mixture. The values of equilibrium constants for both self- and heteroassociation may be determined with reasonable precision from the analysis of data obtained from a single experiment requiring <15 min and <1 mg of each protein. 2), Analysis of the results of a single CG-SLS experiment carried out on Ftsz, a protein that self-associates to form linear oligomers of indefinite size in the presence of guanosine diphosphate, yields the dependence of the equilibrium constant for monomer addition upon oligomer size.

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Infrared spectra of hen egg white lysozyme and bovine serum albumin (BSA) adsorbed on a solid poly tris(trimethylsiloxy)silylstyrene (pTSS) surface in D2O solution were measured using attenuated total reflection (ATR) Fourier transform infrared spectroscopy. From the area and shape of the amide I' band of each spectrum, the adsorption amount and the secondary structure were determined simultaneously, as a function of adsorption time. We could show that the average conformation for all the adsorbed lysozyme molecules was solely determined by the adsorption time, and independent of the bulk concentration, while the adsorption amount increased with the bulk concentration as well as the adsorption time. These results suggest that lysozyme molecules form discrete assemblies on the surface, and that the surface assemblies grow over several hours to have a definite architecture independent of the adsorption amount. As for BSA, the extent of the conformational change was solely determined by the adsorption amount, regardless of the bulk concentration and the adsorption time. These differences in the adsorption properties of lysozyme and BSA may reflect differences in their conformational stabilities.

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Zirconium oxy-salts were hydrolyzed to form positively charged polymer or cluster species in acidic solutions. The zirconium hydrolyzed polymer was found to react with a negatively charged polyelectrolyte, such as poly(vinyl sulfate), and to form a stoichiometric polyion complex. Thus, colloidal titration with poly(vinyl sulfate) was applied to measure the zirconium concentration in an acidic solution by using a Toluidine Blue selective plasticized poly(vinyl chloride) membrane electrode as a potentiometric end-point detecting device. The determination could be performed with 1% of the relative standard deviation. The colloidal titration stoichiometry at pH < or = 2 was one mol of zirconium per equivalent mol of poly(vinyl sulfate).

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The adsorption of hen egg white lysozyme onto a solid polytris(trimethylsiloxy)silylstyrene (pTSS) surface from a D(2)O solution at pD 7 containing 100 mM NaCl and 10 mM sodium deuterated phosphate was monitored at 25 degrees C by Fourier transform infrared spectroscopy using the attenuated total reflection (ATR) method. The infrared spectrum attributed to only the adsorbed lysozyme was derived from the observed spectrum, and the amount of adsorbed lysozyme was determined as a function of time and lysozyme concentration. The kinetics of adsorption could be decomposed into two components, one of which was a process with a time constant of larger than 4 h(-1) and the other was a process with one of about 0.1 h(-1). These spectra showed that the lysozyme adsorbed in the faster process had a higher beta-structure content than the dissolved lysozyme. It was also found that the slower adsorption induced some conformational change in the lysozyme adsorbed in the faster process and/or that adsorbed in the slower process. After adsorption for 24 h, the pTSS surface was rinsed out with lysozyme-free solution. The resultant spectra of the surface indicated that the lysozyme adsorbed in the faster process was bound irreversibly on the surface and was changed to a conformer with a higher beta-structure content during the slower process. The experimental procedures and the theoretical applications for such a quantitative analysis in the ATR spectroscopic method are presented in detail.

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Microsomal aldehyde dehydrogenase (msALDH) is a tail-anchored protein localized to the cytoplasmic face of the endoplasmic reticulum (ER). The carboxyl-terminal 35 amino acids of msALDH possess ER-targeting sequences in addition to a hydrophobic membrane-spanning domain. To study the mechanism for ER targeting of this protein in vivo, we took advantage of a green fluorescent protein-msALDH fusion protein containing the last 35 amino acids of msALDH [GFPALDH(35)]. When expressed from cDNA in COS-7 cells, the fusion protein was localized to the ER. We then prepared a recombinant fusion protein and injected it into the cytoplasm of COS-7 cells. The injected protein was correctly localized to the ER after a 30-min incubation at 37 degrees C. However, a recombinant fusion protein that contained only the transmembrane domain of msALDH failed to be targeted to the ER. When the assay was carried out at 4 degrees C, the recombinant GFPALDH(35) remained in the cytoplasm. Moreover, incubation of COS-7 cells under conditions of ATP depletion resulted in the cytoplasmic distribution of the injected protein. These results indicate that GFPALDH(35) is targeted to the ER post-translationally via an ATP-dependent pathway. This microinjection system worked effectively in different mammalian cell types, suggesting a common mechanism for ER targeting of the tail-anchored protein.

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