Low Mol Fraction Research Articles

Ethylene Glycol Dicyclopentenyl (Meth)Acrylate Homo and Block Copolymers via Nitroxide Mediated Polymerization.

Nitroxide-mediated polymerization (NMP), (homo and block copolymerization with styrene (S) and butyl methacrylate/S) of ethylene glycol dicyclopentenyl ether (meth)acrylates (EGDEA and EGDEMA) was studied using BlocBuilder alkoxyamines. EGDEA homopolymerization was not well-controlled, independent of temperature (90–120 °C), or additional free nitroxide (0–10 mol%) used. Number average molecular weights (Mn) achieved for poly(EGDEA) were 4.0–9.5 kg mol−1 and were accompanied by high dispersity (Ð = Mw/Mn = 1.62–2.09). Re-initiation and chain extension of the poly(EGDEA) chains with styrene (S) indicated some block copolymer formation, but a high fraction of chains were terminated irreversibly. EGDEA-stat-S statistical copolymerizations with a low mol fraction S in initial feed, fS,0 = 0.05, were slightly better controlled compared to poly(EGDEA) homopolymerizations (Ð was reduced to 1.44 compared to 1.62 at similar conditions). EGDEMA, in contrast, was successfully polymerized using a small fraction of S (fS,0 ~ 10 mol%) to high conversion (72%) to form well-defined EGDEMA-rich random copolymer (molar composition = FEGDEMA = 0.87) of Mn = 14.3 kg mol−1 and Ð = 1.38. EGDEMA-rich compositions were also polymerized with the unimolecular succinimidyl ester form of BlocBuilder initiator, NHS-BlocBuilder with similar results, although Ðs were higher ~1.6. Chain extensions resulted in monomodal shifts to higher molecular weights, indicating good chain end fidelity.

Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and iPLA2, but not for Lp-PLA2. Our results also show that none of these PLA2s have significant PLA1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA2, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA2 assays. The sPLA2s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA2 and cPLA2, and activates sPLA2 at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA2 activity and the ability to assay the various forms of PLA2 is important in understanding its function.

A New High-Temperature Transition of Crystalline Cholesterol in Mixtures with Phosphatidylserine

Phosphatidylserine and cholesterol are two major components of the cytoplasmic leaflet of the plasma membrane. The arrangement of cholesterol is markedly affected by the presence of phosphatidylserine in model membranes. At relatively low mol fractions of cholesterol in phosphatidylserine, compared with other phospholipids, cholesterol crystallites are formed that exhibit both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic transition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96°C by differential scanning calorimetry (DSC), using a heating scan rate of 2°C/min. Observation of the transition requires that the hydrated lipid mixture be incubated for several days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96°C is more rapid at higher incubation temperatures. At 37°C the half-time of conversion is ∼7 days. Concomitant with the appearance of the 96°C peak the previously known transitions of cholesterol, occurring at ∼38°C and 75°C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75°C peak takes a longer time than that of the 38°C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75°C and subsequently is converted by additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96°C. After several weeks of incubation at 37°C, only the form with a phase transition at 96°C remains. If such a sample undergoes several successive heating and cooling cycles, the 96°C peak disappears and the 38°C transition reappears on heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of 1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of cholesterol between 0.4 and 0.7, the 38°C transition that reappears after the melting of the 96°C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96°C transition is partially reversible on cooling and reheating. This transition is also scan rate dependent, indicating that it is, at least in part, kinetically determined. The enthalpy of the 96°C transition, after incubation of the sample for 3 weeks at 37°C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with the enthalpy per mole cholesterol increasing between cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar ratio with cholesterol, after incubation at 37°C, exhibits a transition at 95°C that reverses on cooling at 44°C, instead of 60°C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with the essential absence of the 96°C transition in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indicates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with the phospholipid and are not simply in the form of pure crystals of cholesterol. These observations are particularly important in view of recent observations of the presence of cholesterol crystals in biological systems.

Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A 2: structure-function studies with vesicle, micelle, and membrane assays

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A 2 (cPLA 2 and iPLA 2). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH 2) 4COCF 3 and Ph(CH 2) 4PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA 2, and both are poorly effective against cPLA 2. Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA 2 inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA 2 inhibitors. Inactivation of cPLA 2 by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A 2 inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

Lateral Organization of Pyrene-labeled Lipids in Bilayers as Determined from the Deviation from Equilibrium between Pyrene Monomers and Excimers

In lipid bilayers, pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner. The aromatic amine N, N-diethylaniline (DEA), which has a high membrane-to-medium partition coefficient, quenches the monomers only, and therefore it is expected that under conditions in which the monomers are in equilibrium with the excimers due to the mass law, the Stern-Volmer coefficient (Ksv) for monomers (M), defined as KM, should be identical to that of the excimer (E), defined as KE, and KE/KM = 1. 0. This is indeed the case for pyrene and pyrene valerate in egg phosphatidylcholine small unilamellar vesicles. However, for pyrene decanoate and pyrene dodecanoate in these vesicles, and for N-[12-(1-pyrenyl)dodecanoyl]sphingosylphosphocholine in a matrix of either N-stearoyl sphingosylphosphocholine or 1-palmitoyl-2-oleoyl phosphatidylcholine, KE < KM. This can be explained either by the existence of (a) two subpopulations of excimers, one in fast equilibrium with the monomers and the other, related to ground-state protoaggregates of pyrene lipids; (b) two monomer subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both quenched by DEA, but only one of which produces excimers. The good agreement between the photophysical processes determined by steady state and time-resolved measurements supports the third explanation for the bilayers containing pyrene phospholipids. It also suggests that the main factors determining the immiscibility of pyrene lipids in phospholipid bilayers are the temperature, the difference in the gel-to-liquid-crystalline phase transition temperature (deltaTm) between the matrix and the pyrene lipid, and the structural differences between the matrix lipid and the pyrene-labeled lipid. These results indicate that the KE/KM ratio can serve as a very sensitive tool to quantify isothermal microscopic immiscibility in membranes. This novel approach has the following advantages: applicability to fluid phase immiscibility, requirement of a relatively low mol fraction of pyrene lipids, and conceivably, applicability to biological membranes.

Physicomechanical properties of poly(methyl methacrylate‐co‐N‐arylmaleimides)

AbstractThe article describes the effect of incorporating low mol fractions of N‐o‐tolymaleimide (MO)/N‐p‐tolymaleimide (MP) in poly(methyl methacrylate) (PMMA) on Tg, thermal behavior, mechanical, and optical properties. The glass transition temperature (Tg) of PMMA increased by 10–12°C upon incorporation of an ∼ 0.026 mol fraction of N‐arylmaleimides. A further increase of N‐arylmaleimide content up to 0.1 mol fraction in the copolymers resulted in a further ∼ 10°C increase in Tg. The tensile strength and % elongation decreased, whereas modulus increased with increasing maieimide content. The solar transmittance and % transmittance at higher wavelengths of the copolymer sheets having a low mol fraction of N‐arylmalemides (i.e., 0.026–0.05) were comparable to PMMA. © 1995 John Wiley &amp; Sons, Inc.

Miscibility, chain packing, and hydration of 1-palmitoyl-2-oleoyl phosphatidylcholine and other lipids in surface phases

The miscibility of 1-palmitoyl-2-oleoyl phosphatidylcholine with triolein, 1,2-diolein, 1,3-diolein, 1(3)-monoolein, oleyl alcohol, methyl oleate, oleic acid, and oleyl cyanide (18:1 lipids) was studied at the argon-water interface. The isothermal phase diagrams for the mixtures at 24 degrees were characterized by two compositional regions. At the limit of miscibility with lower mol fractions of 18:1 lipid, the surface pressure was composition-independent, but above a mixture-specific stoichiometry, surface pressure at the limit of miscibility was composition-dependent. From the two-dimensional phase rule, it was determined that at low mol fractions of 18:1 lipids, the surface consisted of phospholipid and a preferred packing array or complex of phospholipid and 18:1 lipid, whereas, above the stoichiometry of the complex, the surface phase consisted of complex and excess 18:1 lipids. In both regions of the phase diagram, mixing along the phase boundary was apparently ideal allowing application of an equation of state described earlier (J. M. Smaby and H. L. Brockman, 1984, Biochemistry, 23:3312-3316). From such analysis, apparent partial molecular areas and hydrations for phospholipid, complex, and 18:1 lipid were obtained. Comparison of these calculated parameters for the complexed and uncomplexed states shows that the aliphatic moieties behave independently of polar head group. The transition of each 18:1 chain to the complexed state involves the loss of about one interfacial water molecule and its corresponding area. For 18:1 lipids with more than one chain another two water molecules per additional chain are present in both states but contribute little to molecular area. In contrast to 18:1 lipids, the phospholipid area and hydration change little upon complexation. The uniformity of chain packing and hydration behavior among 18:1 lipid species contrasts with complex stoichiometries that vary from 0.04 to 0.65. This suggests that the stoichiometry of the preferred packing array is determined by interactions involving the more polar moieties of the 18:1 lipids and the phospholipid.

Studies on lysophospholipases: VI. The action of two purified lysophospholipases from beef liver on membrane-bound lysophosphatidylcholine

The action of two lysophospholipases purified from beef liver on lysophosphatidylcholine in microsomal membranes has been studied. Enzyme I, which has been shown to be localized in the soluble fraction of the beef liver cell, has a higher specific activity on microsomal lysophosphatidylcholine than Enzyme II, which originates from the microsomal cell fraction. This trend is also observed with phosphatidylcholine liposomes and single bilayer vesicles in which lysophosphatidylcholine has been incorporated. At low mol fractions of lysophosphatidylcholine in liposomes, the maximum enzymatic rate is proportional to this mol fraction. Similar results are obtained with mixed micelles of lysophosphatidylcholine and Triton X-100. The results are explained in terms of a model in which the two-dimensional substrate density in the membrane surface controls the rate of enzyme action.

A new approach to ion exchange polymers

The observed abnormal behaviour of the electrolyte uptake in ion exchangers (see preceding paper by Glueckauf &amp; Watts) is interpreted quantitatively on the basis of a widespread non-uniformity of distribution of the counter-ions. In the case fully treated in this paper, i.e. for an exchanger of mean molality of 4·17, the range of counter-ion concentrations is shown to extend from about 15 molal to 10 -4 molal. This non-uniformity must have significant effects on the variation of the ion exchange separation factor at different mol-fractions in the exchanger phase. It explains qualitatively the observed fact, that the separation factor is usually larger when the more strongly sorbed ion is present at low mol-fractions, and that this effect increases both with the mean molality of the exchanger, and with the magnitude of the separation factor. The wide non-uniformity also explains the observed variation of the diffusion coefficient of imbibed electrolyte with concentration. A new quantitative treatment is introduced for diffusion through widely non-uniform media. Full agreement between theory and experiment is obtained at high uptake concentrations. At low uptake concentrations, two curves are obtained from the theory, according to whether the low concentration regions in the exchanger are considered as ‘continuous’ or ‘disperse’. The observed data lying between these two calculated curves suggest a considerable ‘continuity’ for the low concentration regions. They indicate the existence of a ‘continuous’ network of submicroscopic aqueous fissures or channels of about 70 Å width, widening occasionally to as much as 2000 Å. The resulting electrical double layer effects in these fissures explain the wide continuous variation of counter-ion concentrations.

Perfect Ternary Solid Solutions

In this paper equations are developed for calculating the solidus and liquidus surfaces, with tie-lines, for ternary systems showing complete solid solubility, based on the assumption that both liquid and solid solutions are perfect. The forms of the liquidus and solidus isotherms predicted by these equations are considered for an hypothetical example. The ternary system of gold, copper and nickel is shown to deviate markedly from ideal behavior at low mol fractions of gold, even though the copper-nickel system is practically ideal in its binary behavior.