James P. Sheridan

Encapsulation of hemoglobin in phospholipid vesicles

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 Å as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4°C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4°C.

Pressure induced changes in liquid alkane chain conformation

Conformations of n‐alkane molecules in the liquid state have been studied as a function of temperature and pressure using Raman scattering. Observations of conformationally sensitive Raman bands in hexane, heptane, octane, and hexadecane reveal no sign of the pressure‐induced effects in polyethylene. Instead the Raman intensities indicate chain kinking as a function of pressure—a dramatic effect in the shorter chains, a slight effect for hexadecane. A theory based on considerations of conformational energy and translational entropy is presented which explains these effects qualitatively.

Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid

A comparative study of the polymorphism exhibited by the polymerizable, tubule-forming phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3- phosphocholine (DC23PC) and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC) in aqueous suspension is reported. Differential scanning calorimetry (DSC), as well as freeze-fracture electron microscopy and Raman spectroscopy, have been used to study the influence on phase behavior of rigid diacetylene groups in the fatty acyl chains of a phosphatidylcholine. DTPC large multilamellar vesicle (MLV) and small unilamellar vesicle (SUV) suspensions were found to retain liposome morphology after chain crystallization had occurred. In marked contrast, diacetylenic DC23PC suspensions do not maintain liposomal morphology in converting to the low temperature phase. Large MLVs of DC23PC with outer diameters in excess of 1 micron convert to a gel phase with cylindrical or tubular morphology at 38 degrees C, just a few degrees below the lipids chain melting temperature (TM(H), i.e. temperature of an endothermic event observed during a heating scan) of 43.1 degrees C. Unlike the large MLVs, small MLVs or SUVs of DC23PC, with diameters of 0.4 +/- 0.3 micron and 0.04 +/- 0.02 micron, respectively, exhibit metastability in the liquid-crystalline state for several tens of degrees below the chain melting temperature prior to converting to a gel phase which, by electron microscopy, manifests itself as extended multilamellar sheets. Raman data collected at TM(H) -40 degrees C demonstrate that the gel state formed by DC23PC is very highly ordered relative to that of DTPC, suggesting that special chain packing requirements are responsible for the novel phase behavior of DC23PC.

Kinetic and thermodynamic studies of the fusion of small unilamellar phospholipid vesicles.

Small phospholipid vesicles, prepared so as to minimize impurities, fuse relatively slowly resulting in the time-dependent development of a characteristic endotherm in differential scanning calorimetry and corresponding changes in the Raman spectrum. The stability of small vesicles towards fusion increases with increasing acyl chain length for the series C-14 through 18. Within the protocols of these experiments, the fusion rate remains unchanged whether the vesicles are held at 10 degrees C below Tm or at Tm itself. We have determined enthalpies of transition for small vesicles and fusion product for C-14 through C-18. In each case delta H for small vesicles is lower than that of the corresponding multilamellar vesicles, while the fusion product delta H is intermediate between small and multilamellar vesicles. The apparent lack of concensus in the literature as to the nature of the fusion process is ascribed to the variety of protocols used as well as the presence or absence of fusion-inducing impurities.

A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes, monoclonal antibodies, and complement

The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate agricultural products and cause intoxication of humans and animals. We have developed a homogeneous competition inhibition assay for T-2 mycotoxin based on complement-mediated lysis of liposomes. The T-2 mycotoxin was converted to an acid chloride derivative, subsequently coupled to the amino group of phosphatidylethanolamine, and incorporated with the phospholipid into unilamellar liposomes. Carboxyfluorescein, which is self-quenched at high concentrations, was entrapped in the liposomes as a release marker. We used a monoclonal IgG1 antibody specific for T-2 mycotoxin and a polyclonal anti-mouse Ig as a secondary antibody since the anti-T-2 IgG1 does not activate complement. In the absence of free T-2, the liposomes were lysed within 30 min after the addition of complement, releasing carboxyfluorescein into the surrounding buffer. In the presence of free T-2 toxin, the binding of antibodies to the liposomes was reduced, causing a corresponding decrease in lysis. This assay proved to be sensitive to T-2 toxin levels as low as 2 ng, which is 10-fold more sensitive than the present enzyme immunoassay using the same antibodies.

Changes in size and shape of liposomes undergoing chain melting transitions as studied by optical microscopy.

Changes in the shape and size of dipalmitoylphosphatidylcholine liposomes at the phase transition at 41.5 degrees C have been monitored by light microscopy. All liposomes change size or shape at the transition and those with simple topologies such as spheres and cylinders can be readily measured. The surface area of these is some 24% greater above the transition than below. This surface area change is virtually identical to that predicted by crystallographic measurements on this system. Also, the rate of transition from one state to another is seen to proceed more rapidly in the smaller liposomes. Optical microscopic observation provides a rapid simple method for monitoring the dependence of the lipid bilayer area on temperature.

Order in Diacetylenic Microstructures

Abstract Polymerizable diacetylenic lipids form a long, hollow tubular bilayer microstructure that has been characterized by microscopy, spectroscopy and x-ray diffraction. The lipid monomers are highly ordered at the molecular level, and at high density the microstructures themselves align giving macroscopic order.

Techniques for Observing Weak Raman Signals in the Diamond Pressure Cell

We wish to report a technique of using the 752.5 nm line of the Kr+ laser to excite the Raman effect in weakly scattering samples in a gasketed diamond anvil pressure cell. The use of this near-infrared excitation substantially reduces the fluorescence of the diamond windows which otherwise tends to distort and overwhelm weak Raman signals. This excitation also reduces interference from the fluorescence of the ruby fragment placed in the cell for pressure measurement. These are, we believe, the first spectra obtained from weak Raman scatterers in a diamond/ruby pressure cell.

Theory of the conformational order of single component sphingomyelin bilayers

Abstract A statistical mechanical analysis of the configurations of the fatty acyl chains of N -lignocerylsphingosinephosphorylcholine (n-C 24 ) and N -palmitoylsphingosinephosphorylcholine (n-C 16 ) is presented for the case of planar multibilayers. The configurations of the chains are constrained to lie on a three-dimensional lattice, and are generated by the three-dimensional paths of a specified unrestricted random walk on a two-dimensional square lattice. The order-disorder phase transition undergone by the fatty acyl chains is analyzed in light of the model. The transition temperature and enthalpies of transition are predicted in good agreement with calorimetric data. The experimental melting curve of (n-C 24 ) as measured by Raman spectroscopy, displays a post-transitional melting phenomenon indicative of excess trans bonds persisting beyond the main transition. This particular thermal behavior, absent in n-C 16 is explained with the aid of our theory. In particular, the theoretically predicted ratios of the high temperature (fluid) to the low temperature (gel) melting curve slopes agree very well with the experimental values for n-C 24 and n-C 16 . The n-C 24 molecules are predicted to be interdigitated in the gel phase. We discuss the use of our model to predict the equilibrium configurations of fatty acyl chains in membranes in the presence of unsaturated carbon-carbon bonds.

Raman Study of the Conformational Characteristics of Chain Molecules under High Pressure

As part of a continuing program to study the properties of lubricants and polymers, we have undertaken an investigation of the conformational properties of linear alkanes and polymers as functions of temperature and pressure. We have successfully obtained the Raman spectra of these materials over a range of 1 to 20 kbar from a gasketed diamond-anvil cell while calibrating the pressure with the ruby fluorescence technique [1]. The conformation of heptane (C7H34) and hexadecane (C16H34) have been monitored by the Observation of conformationally sensitive Raman bands. Our data indicate that heptane becomes more “kinked” (more gauche bonds) as a function of pressure. This result was surprising to us at first, since at sufficiently high pressure heptane freezes in the unkinked, all-trans conformation. We have developed a theory, based on excluded volume considerations, which is consistent with these observations.