气/液界面及固体表面硬脂酸LB膜结构性质研究

对气液/和固/液界面上硬脂酸LB膜的结构性质的研究表明,二价离子能够使气/液界面上LB膜表面压力降低,并出现一个固-固转变的过程.对此可以解释为是由二价离子富集在亚相表面,减弱了膜分子之间的库仑作用,使表面电势降低引起的.同时由于二价离子与硬脂酸分子形成复合物,单层膜的结构发生改变,导致固-固转变点的产生.对固体基质上多层LB膜的椭圆偏振研究表明,有序排列的硬脂酸LB膜具有明显的双折射性质.电镜观察发现两个固相垂直提位获得的多层膜在形貌上存在差异,低压固相膜较之高压固相膜存在明显的不均匀性.分析认为这是在膜从气/液界面向固体表面转移过程中发生重结晶引起的.     

Polynucleotide adsorption to negatively charged surfaces in divalent salt solutions

Polynucleotide adsorption to negatively charged surfaces via divalent ions is extensively used in the study of biological systems. We analyze here the adsorption mechanism via a self-consistent mean-field model that includes the pH effect on the surface-charge density and the interactions between divalent ions and surface groups. The adsorption is driven by the cooperative effect of divalent metal ion condensation along polynucleotides and their reaction with the surface groups. Although the apparent reaction constants are enhanced by the presence of polynucleotides, the difference between reaction constants of different divalent ions at the ideal condition explains why not all divalent cations mediate DNA adsorption onto anionic surfaces. Calculated divalent salt concentration and pH value variations on polynucleotide adsorption are consistent with atomic force microscope results. Here we use long-period x-ray standing waves to study the adsorption of mercurated-polyuridylic acid in a ZnCl2 aqueous solution onto a negatively charged hydroxyl-terminated silica surface. These in situ x-ray measurements, which simultaneously reveal the Hg and Zn distribution profiles along the surface normal direction, are in good agreement with our model. The model also provides the effects of polyelectrolyte line-charge density and monovalent salt on adsorption.     

Effects of polyamines on the activities of Escherichia coli ribonuclease I and II.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by E. coli ribonuclease I [ribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.23] and ribonuclease II [EC 3.1.4.1] have been studied. The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines. Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines. The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations. When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence. Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)greater than poly(A)(see text)poly(U). However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased.     

Polyelectrolyte properties of biopolymers: conductivity and secondary structure of polyriboadenylic acid and its salts in solutions

Polyriboadenylates of alkali metals were obtained from (1) K(+)-poly(A) (salts 1) and (2) H(+)-poly(A) (salts II) by the ion-exchange method. The conductivity of these salts as well as of H(+)-poly(A) were studied. Salts I and II of the same counterion were shown to have significantly different conductivity coefficients (f) and polyion conductances (lambda 0p). the charge density parameter (xi) was 1.3 and 2.5, respectively, with lambda 0p equal to 44 and 83 ohm-1 cm2 mole-1 for poly(A)-I and poly(A)-II salts, respectively. This is credited to the difference in the conformations of corresponding polyions. The linear dependence of equivalent conductivity on the square root of polymer concentration (Kohlrausch coordinates), earlier obtained for DNA, is also satisfied for the studied polynucleotides. A comparison of the slopes of straight lines in Kohlrausch coordinates for poly(A), simple electrolytes, and for earlier studied polyribouridylic acid salts lends credence to the concepts, developed by a number of authors, that DNA can act as a "buffer" against the ion-ion interaction in concentrated electrolyte solutions. Using the approximation that the polyion conductance is independent of the counterion nature, parameter f (agreeing in this case with Eisenberg parameter phi) has been shown to decrease as the polynucleotide concentration is increased; the decrease is caused by the relaxation effect. The transference numbers of counterions, which have negative values in poly (A)-II solutions, grow with the increase in polymer concentration; the higher the xi, the more apparent is this increase. This is explained by the increase in the fraction of conductivity along the polyion chains ("surface" conductivity) with the growth of polyelectrolyte concentration.     

Surface properties of two rabbit lung lamellar body preparations with markedly different fatty acid profiles

The effect of fatty acid desaturation on the surface properties of lung surfactant were studied on a Wilhelmy surface balance by using two preparations of lamellar body (LB) material with markedly different fatty acid profiles: (1) lamellar bodies from adult rabbit lung tissue, and (2) lamellar bodies from fetal rabbit lung tissue maintained in organ culture for 7 days. The fetal lung preparation contains an unusually high level of 16: 1 fatty acid (principally palmitoleic acid) at position sn-2 of phosphatidylcholine (Longmuir, K.J., Resele-Tiden, C. and Rossi M.E. (1988) J. Lipid Res. 29, 1065-1077). Surface pressure-surface area isotherms were obtained for both preparations and compared to isotherms of monolayers of dipalmitoylphosphatidylcholine. In addition, the elasticity of the lamellar body preparations were analyzed as a function of surface pressure, temperature, and rate of compression, both in the presence and absence of Ca2+ plus Mg2+. At slow rates of compression, we found that fetal LB films have lower elasticity and better respreading ability compared to the adult LB films, which can be explained by the high concentration of unsaturated palmitoleic acid in the fetal preparation. A dynamic component of elasticity was observed at high rates of compression only if Ca2+ and Mg2+ were present in the subphase. The analysis of the free energies, enthalpies and entropies of compression suggests that films with low concentrations of unsaturated fatty acids are are likely to undergo irreversible collapse, but films with excess unsaturated fatty acids accommodate the overcompression with a reversible loss of molecules from the surface.     

STM studies of LB films of amphiphilic polyelectrolytes with antibodies and enzymes

The structure of LB films of protein-polyelectrolyte complexes transferred onto the pyrographite surface was studied by STM. The images of the films obtained at various protein concentrations in the water subphase and different values of surface pressure were captured. The topology of the surface covered by one or three layers of the films studied was investigated. It is shown that at a protein concentration of 1 mg/ml in the water subphase, the films are composed of aggregated protein molecules, and their structure has an insular character. An increase in the number of transferred layers up to three results in a virtually complete covering of the surface. A decrease in the protein concentration in the water subphase to 1 microgram/ml enabled us to prepare films consisting of individual non-aggregated protein molecules.     

Interaction of natural and synthetic polynucleotides with liposomes in the presence of divalent cations

The formation of complexes of polynucleotides (DNA, poly A.poly U) with liposomes from egg lecithins, L-alpha-phosphatidylcholine, dimirystoyl and other lipids in the presence of divalent cations was studied by differential scanning microcalorimetry circular dichroism and turbidimetry. It was shown that the secondary structure of polynucleotides (double or triple helix) was necessary for the formation of these complexes. This structure was partially destroyed during formation of complexes. It was shown, that three main types of lipids, i.e. phosphatidylcholine, phosphatidylethanolamine and sphingomyelin participate in interactions between liposomes, polynucleotides and Mg2+.     

Hydrogen bonding versus ion pairing in polyelectrolyte multilayers with homopolynucleotides

Homopolynucleotides--poly(adenylic acid), poly(A), and poly(uridylic acid), poly(U)--were assembled, layer-by-layer, into thin films with poly(ethylenimine), PEI. Various combinations and sequences of polynucleotide and PEI were used to highlight contributions of electrostatic versus hydrogen bonding as driving forces for multilayer build-up. Assembly of alternating poly(A) and poly(U) failed to yield growing films, due to excessively strong interactions between these complimentary strands. The surface morphology of multilayers depended on the deposition order and whether films had been annealed by salt. Films assembled from preformed A/U duplexes (having high persistence lengths) were very smooth. Individual adsorption steps, followed by optical waveguide light-mode spectroscopy, showed that only complementary polynucleotides adsorb by H-bonding to the surface of a growing multilayer. In contrast to behavior usually observed for polyelectrolyte multilayer build-up, the films decreased in thickness with increasing salt concentration.     

Interaction of metal ions with polynucleotides and related compounds. IX. Unwinding and rewinding of poly(A + U) and poly(I + C) by copper(II) ions

It is demonstrated that, poly(A + U) and poly(I + C) are both formed under low ionic strength conditions. Continuous variation studies indicate the formation of copper(II) complexes of poly A, poly C, and poly I, but not of poly U. Copper(II) in a 1:1 ratio to polynucleotide prevents the formation of poly(A + U) and brings about the dissociation of the poly (A + U) complex produced in the absence of the metal. Poly (I + C) is similarly dissociated by copper(II) ions. The addition of sufficient electrolyte reverses the copper(II) induced dissociation of poly(I + C). The effect of copper(II) on ordered synthetic polynucleotides is thus very similar to its effect on DNA.     

Potentiometric titration of poly(L-glutamic acid) in aqueous solutions and binding of divalent cations

The potentiometric titration of poly(L -glutamic acid) was performed under conditions of varied ionic strength and concentration of added divalent cations. From these titration curves, the amount of divalent cations, especially magnesium, bound to poly(L -glutamic acid) was determined using a new method of analysis based on polyelectrolyte theory. By comparison with the polyelectrolyte, poly(acrylic acid), it was found that there are no specific interactions between metal ion and poly(L -glutamic acid) in either the helical or random coil conformation. The effect of these divalent cations on the conformation of poly(L -glutamic acid) was also discussed.     

Purification and characterization of a major endonuclease from rat liver nuclei.

A major endonuclease has been purified approximately 800-fold from rat liver nuclei using poly(A) as substrate. The enzyme had a molecular weight of about 50,000, and active fractions were obtained which contained no nucleic acid. Enzymatic activity was optimal between pH 6 and 7 and was totally dependent on the presence of a divalent cation. The reaction was inhibited by high ionic strength, polydextran sulfate, heparin, and sodium pyrophosphate. The purified enzyme readily hydrolyzed poly(A), poly(U), poly(C), and denatured DNA, whereas poly(G) was not degraded, and transfer RNA, ribosomal RNA, and native DNA were hydrolyzed only at relatively slow rates. These data suggest that the enzyme may be specific for single-stranded polynucleotides. The purified enzyme was essentially devoid of exonuclease activity, and the products of exhaustive endonuclease digestion of poly(A) were small oligonucleotides terminated with a 5'-phosphoryl group. Evidence was obtained that this endonuclease is localized in the nucleoplasm. Possible functions for this activity are discussed.     

Influence of stearic acid monolayers upon the procaine adsorption from underlying alkaline aqueous solutions

Adsorption of procaine at the air/water interface and its penetration into stearic acid monolayers from aqueous subphase of pH 8 are studied by measuring surface tension of aqueous procaine solutions and by recording surface pressure vs. mean molecular area curves for stearic acid monolayers spread onto procaine solutions of different concentrations. The amount of procaine in the interface is derived by means of Gibbs' equation. Results are compared to those obtained earlier at pH 2 and on unbuffered subphases. With increasing pH an increasing procaine adsorption and procaine penetration is observed. This phenomenon is interpreted in terms of protolytic equilibria in which participate both surfactants procaine and stearic acid.     

Langmuir and Langmuir-Blodgett films of organophosphorus acid anhydrolase

In this paper, we describe the preparation and characterization of Langmuir and Langmuir-Blodgett (LB) monolayers of the enzyme organophosphorus acid anhydrolase (OPAA). Langmuir films of OPAA were characterized on different subphases, such as phosphate, ammonium carbonate, and bis-tris-propane buffers. Monolayers at the air-water interface were characterized by measuring the surface pressure and surface potential-area isotherms. In situ UV-vis absorption spectra were also recorded from the Langmuir monolayers. The enzyme activity at the air-water interface was tested by the addition of diisopropylfluorophosphate (DFP) to the subphase. LB films of OPAA were transferred to mica substrates to be studied by atomic force microscopy. Finally, a one-layer LB film of OPAA labeled with a fluorescent probe, fluorescein isothiocyanate (FITC), was deposited onto a quartz slide to be tested as sensor for DFP. The clear, pronounced response and the stability of the LB film as a DFP sensor show the potential of this system as a biosensor.     

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions

Owing to its distinct chemico-biological properties, chitosan, a cationic biopolymer, offers a great potential in multifarious bioapplications. One such application is as a dietary antilipidemic supplement to be used to reduce obesity/overweight and to lower cholesterol. The lipid-binding efficiency of chitosan, however, remains debatable. Accordingly, in this study we investigated the interactions of chitosan with selected lipids, cholesterol and fatty acids, the latter including saturated (stearic acid) and unsaturated (oleic, linoleic, alpha-linolenic) acids. The experiments were performed with the Langmuir monolayer technique, in which surface pressure-area isotherms were recorded for the lipid monolayers spread on the acetate buffer pH 4.0 subphase in the absence and presence of chitosan. We found that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and alpha-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results we tentatively propose a mechanism of the chitosan action that includes both electrostatic and hydrophobic lipid-chitosan interactions as well as hydrogen bonding between them.     

Direct electrochemistry of hemoglobin immobilized on gold electrode by Langmuir-Blodgett technique

In this research, we reported a novel method of forming hemoglobin (Hb)-linoleic acid (LA) Langmuir-Blodgett (LB) monolayer by spreading Hb solution directly onto the subphase covered with a layer of LA. This method is suitable for preparing electrochemical devices with protein-lipid LB film because almost no protein adsorbed on electrode surface before protein-lipid film transferred from air-water interface to electrode, which ensured better electrode activity. The compressibility of Hb-LA monolayer was used to character the phase transition during compression process. Optimal experimental conditions were obtained by analyzing pressure-time, pressure-area and pressure-compressibility curves. The direct electrochemistry of Hb, which was immobilized on Au electrode surface incorporated with LA layer by LB method, was investigated using cyclic voltammetry for the first time. The electrode modified with Hb-LA LB film holds high electrochemical activity and shows a fast direct electron transfer of Hb. Redox peak currents increased linearly with the increase of scan rate, indicating a surface-controlled electrode process. The electron transfer rate constant was 2.68+/-0.45 s-1. As a target of this research, this work provides a new way to prepare biomimetic film and biosensor.     

Effects of polyamines on the degradation of ribonucleic acids by polynucleotide phosphorylase of Micrococcus luteus.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C) greater than poly(A) greater than poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [14C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.     

Anti-complement activity of polynucleotides.

A biologic assay system, based on complement (C′) inhibition, is described to unravel structural differences among polynucleotides. The C′ system appears particularly suitable to distinguish (1) homo- from copoly-ribonucleotides, (2) deoxyribo- from 2′-OH and other 2′-modified polynucleotides, and (3) single homopolynucleotides from double- or triple-stranded complexes.From these studies a number of polynucleotides emerged with potent anti-C′ activities, worthy of further investigation. The most active polymers were (G)_n (polyguanylic acid), (dCc1)_n [poly(2′-chloro-2′deoxycytidylic acid)] and (dUz)_n [poly(2′-azido-2′-deoxyuridylic acid)].     

Carcinogenic purine N-oxide ester modifies covalently all common bases in polynucleotides

The carcinogen 1-methyl-3-hydroxyxanthine after esterification binds covalently to polynucleotides, RNA and DNA. All four ribopolynucleotides and poly(dT) are targets. Depending on reaction conditions, covalent binding is greatest to poly(A) followed by poly(U), poly(dT), poly(G), poly(C), RNA and DNA. Maximal covalent modification of DNA is one moiety per 360 nucleotides. All modified polynucleotides, RNA and DNA, except poly guanylic acid have been enzymatically digested and the major adducts characterized as nucleosides.     

Chitosan-cholesterol and chitosan-stearic acid interactions at the air-water interface

We report in this work the isotherms of cholesterol and stearic acid at the air-water interface modified by different chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weight chitosans) in the aqueous subphase. The Langmuir-Blodgett films of the complexes cholesterol-chitosan and stearic acid-chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulation was performed to visualize the chitosan-lipid interactions. Strong modifications are obtained in the isotherms as a result of the chitosan interactions with cholesterol and stearic acid at the air-water interface. These modifications were dependent on the type and concentration of chitosan. Severe modifications of all phases were noticed with larger molecular areas, and the observed changes in the compressional modulus were dependent on the type of chitosan used. The complexes of chitosan-stearic acid were more flexible than the ones of chitosan-cholesterol. The AFM images demonstrated that chitosan was disaggregated by the cholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. The hydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weight chitosans produced homogeneous surfaces with cholesterol. The simulated chitosan chains interacting with cholesterol and stearic acid demonstrated the possibility of specific sites of electrostatic bonds between these molecules. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showed that the medium and high molecular weight chitosans could retain higher proportions of cholesterol compared with the other analyzed samples.     

The binding of spermine to polynucleotides and complementary oligonucleotides at near physiological ionic strength

The binding of [14C] spermine to polynucleotides has been studied by equilibrium dialysis and the data analysed by Scatchard plots. The binding of spermine to poly(A) shows a binding site for 1 spermine/140 nucleotides when measured in 0.2M NaCl at 5 degrees C. Poly(C) also has a similar sites; on the other hand poly(U) and poly(G) each have a binding site for 1 spermine/12 nucleotides. The addition of complementary di- or trinucleotides to either poly(A) or poly(U) affects their ability to bind spermine, in particular the high affinity site on poly(A) is no longer detectable. The effect of spermine, spermidine and putrescine on the binding of polynucleotides to complementary di- and trinucleotides was also studied. Spermine markedly increased the binding of both ApA and of ApApA to poly(U) whereas spermidine and putrescine had very little effect. In contrast spermine had little effect on the binding of either UpU or UpUpU to poly(A). These results suggest that spermine binding to oligo- and polynucleotides is dependent on the particular nucleotide combination involved and that spermine may therefore be able to act selectively within cells.