Bruce P. Gaber

Interpretation of biomembrane structure by Raman difference spectroscopy. Nature of the endothermic transitions in phosphatidylcholines.

Raman difference spectroscopy has been applied to aqueous dispersions of dipalmitoyl phosphatidylcholine (DPPC). Difference spectra have been created by computer subtraction of absolute Raman spectra taken in each of three different temperature ranges: below the endothermic pretransition at 34 +/- 2 degrees C; between this temperature and the melting transition at 42 degrees C; and above the melting temperature. The resultant difference spectra are both quantitatively and qualitatively different, indicating that a distinct phospholipid conformation occurs in each of the three temperature ranges. Furthermore, the difference spectra show details of Raman spectral changes with greater clarity than is possible with conventional Raman techniques. A description of the lateral interchain order and the longitudinal chain order is given for each of the three temperature ranges. In addition to obtaining a more precise quantitative measurement of the changes in the Raman spectra, we observed some significant and previously unreported changes. It is suggested that distortion in the hexagonal lattice below the pretransition temperature previously reported by X-ray diffraction techniques may be responsible for interchain interactions which give rise to a Raman band observed only in the triclinic lattice of even-numbered n-alkanes.

Thin film nanofabrication via layer-by-layer adsorption of tubule halloysite, spherical silica, proteins and polycations

Abstract The microcylinders of halloysite of 50 nm diameter, 500 nm length and with 20 nm diameter hollow inner core were used in the assembly by alternate adsorption with poly(ethyleneimine) (PEI), which resulted in the formation of ordered multilayers containing from 2 up to 20 layers of the tubules glued together by polycation interlayers. The tubules in each monolayer are loosely packed; they form a network in the remaining 50% empty space. The total thickness of the (halloysite/PEI) 14 film was found to be 720 nm, i.e. 54 nm for the layer pair. The tubule/sphere superlattice films were also assembled through alternation of halloysite, 45-nm diameter silica spheres and linear polycations. An assembly of alcohol dehydrogenase (ADH) and halloysite loaded with nicotinamide adenine dinucleotide in alternation with PEI was accomplished. This assembly is designed to provide a direct supply of the ADH-cofactor to the enzyme immobilized in polyion films.

Effects of chain packing and chain mobility on the Raman spectra of biomembranes.

The degree of lateral crystal-like order between hydrocarbon chains in biomembrane systems can be estimated from Raman measurements in the C-H stretching region. Observations of the temperature dependence of the Raman spectra of crystalline n-C16H34 and the urea clathrate of n-C16H34 have enabled us to separate to some extent the overlapping effects of chain packing and chain mobility, effects that are normally not distinguished in considering lateral order. The mobility is associated with the freedom of an extended chain to rotate and twist about its long axis. A high degree of such motion must be ascribed to n-C16H34 in a urea clathrate in order to explain the unusual temperature behavior observed for Raman bands at 2885 and 1174 cm-1. Comparison of the temperature behavior of the Raman spectra of the clathrate with that of crystalline n-C16H34 permits the effects due to packing and to mobility to be distinguished. The same effects can be expected to be present in the Raman spectra of biomembranes.

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 studies on two hydrated phospholipids — 1,2-dimyristoyl-phosphatidylcholine and 1,2-dipalmitoyl-phosphatidylcholine

We present results of studies on the effect of pressure on phase transitions in 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-phosphatidylcholine (DPPC) dispersed in excess water. The P-T diagram of hydrated DMPC shows a Gel III-Gel II-Gel I triple point at 3.5 kbar, 41 degrees C, the Gel III phase being obtained by annealing the sample at high pressure for several hours. In the case of DPPC, a pressure induced phase (X) appears between the Gel II and Gel I phases at approximately 0.93 kbar. With increasing pressure the temperature range of the X phase increases at the expense of that of the Gel I phase until finally at 2.87 kbar, the latter is completely suppressed. The P-T diagram of water-rich DPPC thus has 2 triple points, the Gel II-X-Gel I triple point at 0.93 kbar, 42.5 degrees C and the X-Gel I-liquid crystal triple point at 2.87 kbar, 98.5 degrees C. A pressure induced Gel III-Gel II transition is also observed in DPPC in the pressure range of 1.7-3 kbar.

Deuterated phospholipids as nonperturbing components for Raman studies of biomembranes.

The deuterated phospholipid, 1,2-dipalmitoyl-d62-phosphatidylcholine is shown by Raman spectroscopic measurements to be useful for obtaining information concerning phospholipid conformation in complex phospholipid and lipidprotein mixtures. The Raman bands of the deuterated phospholipid are assigned, and the sensitivity of these vibrational modes to conformational changes in the bilayer is demonstrated. Deuteration of the alkyl chains reveals the CH vibrations of the head group. A change in these bands is observed at the melting temperature and is assigned to alteration of the glycerol backbone conformation upon melting.

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.

Control of surface expression of functional groups on silica particles

The effect of the time of addition of organosilanes to silica particle formation reaction mixtures on the resulting surface availability of the added functional groups was investigated. Base catalyzed particle formation was initiated by the addition of tetraethyl orthosilicate (TEOS) to a water-in-oil microemulsion. Subsequently, amine, mono-carboxylate, ethylenediaminetriacetic acid, or dihydroimidazole-terminated organosilanes were added to the microemulsion. Continuous growth in size of monodispersed spherical particles over time was monitored by transmission electron microscopy and light-scattering measurements. Surface primary amine and carboxylate groups on the resulting particles were labeled with fluorescamine and 1-pyrenyldiazomethane (PDAM), respectively, and the effect of varying the time of organosilane addition to the microemulsion was determined by fluorescence spectroscopy. The effect of the time of organosilane addition on surface expression of dihydroimidazole groups was determined using a direct titration method. The results demonstrate that the degree of surface functionalization of the silica particles varied with the time of organosilane addition. For each type of amine terminated organosilane used, the highest surface availability of organo-functional groups was obtained when the organosilane was added 30 min after particle growth was initialized. A progressive decrease in the surface availability of primary amine groups was observed when 3-aminopropyltrimethoxysilane (APTMS) was added later than 30 min after particle formation was initiated. For each type of carboxylate-terminated organosilane used, the highest surface availability of organo-functional groups was obtained when the organosilane was added 5.5 h after particle growth was initialized.

Molecular modelling of saccharide-lipid interactions

Certain saccharides, including trehalose, sucrose and glucose, stabilize lipid bilayers against dehydration. It has been suggested that these saccharides replace waters of hydration as the system is dried, thereby maintaining the headgroups at their hydrated spacing. The lipid acyl chains consequently have sufficient free volume to remain in the liquid crystallines state, and the processes that disrupt membrane integrity are inhibited. Initial molecular graphic investigations of a model trehalose/DMPC system supported this idea (Chandrasekhar, I. and Gaber, B.P. (1988) J. Biomol. Stereodyn, 5, 1163–1171). We have extended these studies to glucose and sucrose. A set of AMBER potential parameters has been established that reproduce simple saccharide conformations, including the anomeric effect. Extensive energy minimizations have been conducted on all three systems. The saccharide-lipid interaction energies become less stable in the order trehalose <sucrose<glucose. However, the total energies of the saccharide/lipid complexes becom eless negative in the order trehalose<glucose<sucrose.