Differential scanning calorimetry and 31P NMR studies on sonicated and unsonicated phosphatidylcholine liposomes.

1. Phase transitions in sonicated (vesicles) and unsonicated liposomes composed of various synthetic phosphatidylcholines are monitored using differential scanning calorimetry and 31P NMR. 2. The temperature (Tc), heat content and width of the phase transition are comparable in both vesicles and liposomes prepared from 1,2-dipalmitoyl phosphatidylcholine and 1,2-dimyristoyl phosphatidylcholine. In vesicles composed of a (1 : 1) mixture of 1,2-dipalmitoyl phosphatidylcholine and 1,2-dioleoyl phosphatidylcholine phase separation occurs as in the bilayers of the unsonicated liposomes. 3. The linewidth of the 31P resonances in vesicles is not greatly dependent upon the fatty acid composition when the lipids are in the disordered liquid crystalline state (above Tc). When the lipids are in the gel state (below Tc), however, there is a marked increase in linewidth, demonstrating a reduction in motion of the phosphate group. 4. The ratio of the amounts of phosphatidylcholine present in the outside and inside monolayter of the vesicle membrane was determined with 31P NMR using Nd3+ as a non-permeating shift reagent. 5. The outside/inside ratio is dependent upon the hydrocarbon chain length. Increasing chain length gives a lower outside/inside ratio and a larger vesicle. Introduction of cis or trans double bonds in the chain influences the outside/inside ratio slightly. 6. The incorporation of cholesterol decreases the outside/inside ratio and increases the size of 1,2-dimyristoyl phosphatidylcholine vesicles. The cholesterol concentration in the outside and inside monolayer is approximately the same. The size of the 1,2-dioleoyl phosphatidylcholine vesicles is also increased by cholesterol incorporation but the outside/inside distribution is also increased, especially between 30 and 50 mol% cholesterol. In these vesicles cholesterol is asymmetrically distributed and strongly prefers the inside monolayer of the vesicle.     

Differential scanning calorimetry studies of rabbit cardiac sarcolemma

Thermal transitions were measured by differential scanning calorimetry for rabbit cardiac sarcolemma in 3-(N-morpholino)propanesulfonic acid buffer at pH 7.5, in glycerol-buffer and dimethyl sulfoxide - buffer mixtures, after heat denaturation, and after enzymatic degradation of the proteins. Specific solvent effects on the protein transitions were observed. Glycerol stabilized some of the four protein transitions, while dimethyl sulfoxide destabilized all protein transitions. The thermal transitions in the lower temperature range were studied for both the membranes and the lipid extracted from the membranes. A very small endotherm was observed for both the lipid extracted from the sarcolemma and the intact membrane (0.1-0.2 cal/g; 1 cal = 4.1868 J). A larger endotherm was observed in both the glycerol-buffer and dimethyl sulfoxide - buffer mixtures. Major perturbation of the protein by enzymatic degradation (papain or trypsin digestion), by heat denaturation, or by reaction with excess N-ethylmaleimide all produced larger endotherms near 20 degrees C. The very small magnitude of the endotherm near 20 degrees C suggests that it is not a typical gel - liquid crystalline transition of the bilayer. However, the occurrence of an endotherm in the extracted lipid suggests that some reorientation of lipid is involved.     

Differential scanning calorimetry on mixtures of lecithin,lysolecithin and cholesterol

The effect of increasing concentrations of lysolecithin (1-palmitoyl-sn-glycerol-3-phosphorylcholine) on the gel → liquid crystal thermal transition of lecithin (1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine) in the aqueous phase was studied by differential scanning calorimetry (DSC). Lysolecithin showed an endothermic transition at 3.4°C whereas the transition of the lecithin occurred at 42°C. No phase separation could be observed calorimetrically at lysolecithin concentrations up to 60 mol%. Freeze etch electron microscopy showed that mixtures containing as much as 50 mol% lysolecithin exist in a lamellar phase. The lysolecithin was found to cause an initial slight increase in the enthalpy of transition followed by a gradual decrease. The enthalpy increased again at very high lysolecithin concentrations. The lysolecithin also caused a non-linear decrease in the temperature at which the lecithin transition took place.Cholesterol was found to decrease the enthalpy of transition of the lysolecithin, eliminating it at a concentration of 50 mol%. Cholesterol caused an increase in the temperature at which the lysolecithin transition took place.     

Differential scanning calorimetry of chain-melting phase transitions of N-acylphosphatidylethanolamines.

Phosphatidylethanolamines in which the polar headgroup is N-acylated by a long-chain fatty acid (N-acyl PEs) are present in many plasma membranes under normal conditions, and their content increases dramatically in response to membrane stress in a variety of organisms. The thermotropic phase behavior of a homologous series of saturated N-acyl PEs, in which the length of the N-acyl chain is equal to that of the O-acyl chains attached at the glycerol backbone, has been investigated by differential scanning calorimetry (DSC). All fully hydrated N-acyl PEs with even chain lengths from C-12 to C-18 exhibit sharp endothermic chain-melting phase transitions in the absence of salt and in 1 M NaCl. Cooperative chain-melting is demonstrated directly by the temperature dependence of the electron spin resonance spectra from probe phospholipids bearing a spin label group in the acyl chain. The calorimetric transition enthalpy and the transition entropy obtained from DSC depend approximately linearly on the chain length with incremental values per CH2 group that exceed those of normal diacyl phosphatidylethanolamines, but to an extent that underrepresents the additional N-acyl chain. A thermodynamic model is constructed for the chain-length dependences and end effects of the calorimetric quantities, which includes a deficit proportional to the difference in O-acyl and N-acyl chain lengths for nonmatched chains, as is found and justified structurally for mixed-chain diacyl phospholipids. From data on the chain-length dependence of N-acyl diC16PEs, it is then deduced that the N-acyl chains are less well packed than the O-acyl chains and, from the data on the matched-chain N-acyl PEs, that the O-acyl chain packing is similar to that in normal diacyl PEs. The gel-to-fluid phase transition temperatures of the N-acyl PEs in the absence of salt are practically the same as those of the normal diacyl PEs of the corresponding chain lengths, although the transition enthalpies and entropies are appreciably greater, indicating entropy-enthalpy compensation. In 1 M NaCl, the transition temperatures are 3-4.5 degrees higher than in the absence of salt, representing the contribution of the electrostatic surface potential of the N-acyl PEs.     

Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry

Deuterium nuclear magnetic resonance spectroscopy and differential scanning calorimetry are used to map the phase boundaries of mixtures of cholesterol and chain-perdeuteriated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine at concentrations from 0 to 25 mol % cholesterol. Three distinct phases can be identified: the L alpha or liquid-crystalline phase, the gel phase, and a high cholesterol concentration phase, which we call the beta phase. The liquid-crystalline phase is characterized by highly flexible phospholipid chains with rapid axially symmetric reorientation; the gel phase has much more rigid lipid chains, and the motions are no longer axially symmetric on the 2H NMR time scale; the beta phase is characterized by highly ordered (rigid) chains and rapid axially symmetric reorientation. In addition, we identify three regions of two-phase coexistence. The first of these is a narrow L alpha/gel-phase coexistence region lying between 0 and about 6 mol % cholesterol at temperatures just below the chain-melting transition of the pure phospholipid/water dispersions, at 37.75 degrees C. The dramatic changes in the 2H NMR line shape which occur on passing through the phase transition are used to map out the boundaries of this narrow two-phase region. The boundaries of the second two-phase region are determined by 2H NMR difference spectroscopy, one boundary lying near 7.5 mol % cholesterol and running from 37 down to at least 30 degrees C; the other boundary lies near 22 mol % cholesterol and covers the same temperature range. Within this region, the gel and beta phases coexist. As the temperature is lowered below about 30 degrees C, the phospholipid motions reach the intermediate time scale regime of 2H NMR so that spectral subtractions become difficult and unreliable. The third two-phase region lies above 37 degrees C, beginning at a eutectic point somewhere between 7.5 and 10 mol % cholesterol and ending at about 20 mol %. In this region, the L alpha and beta phases are in equilibrium. The boundaries for this region are inferred from differential scanning calorimetry traces, for the boundary between the L alpha- and the two-phase region, and from a dramatic sharpening of the NMR peaks on crossing the boundary between the two-phase region and the beta-phase region. In this region, the technique of difference spectroscopy fails, presumably because the diffusion rate in both the L alpha- and beta-phase domains is so rapid that phospholipid molecules exchange rapidly between domains on the experimental time scale.     

1H NMR relaxation studies of protein-polysaccharide mixtures

NMR water proton relaxation was used to characterize the structure of plant proteins and plant protein-polysaccharide mixtures in aqueous solutions. The method is based on the mobility determination of the water molecules in the biopolymer environment in solutions through relaxation time measurements. Differences of conformation between pea globulin and alpha gliadin seem to control the water molecules mobility in their environment. As deduced from the study of complexes, the electrostatic interactions may also play a major role in the water molecule motions. The phase separation induced under specific conditions seems to promote the translational diffusion of structured water molecules whereas the rotational motion was more restricted.     

Differential scanning calorimetry of lobster haemocyanin

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxima is found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two-state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing omega-cyclohexyl fatty acids. Differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of aqueous dispersions of 10 phosphatidylcholines containing omega-cyclohexyl-substituted acyl chains was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The presence of the omega-cyclohexyl group has a profound effect on the thermotropic phase behavior of these compounds in a manner dependent on whether the fatty acyl chains have odd- or even-numbered linear carbon segments. The thermotropic phase behavior of the odd-numbered phosphatidylcholines is characterized by a single heating endotherm that was shown to be a superposition of at least two structural events by calorimetric cooling experiments. 31P NMR spectroscopy also showed that the single endotherm of the odd-chain compounds is the structural equivalent of a concomitant gel-gel and gel to liquid-crystalline phase transition. The calorimetric behavior of the even-numbered phosphatidylcholines is characterized by a complex array of gel-state phenomena, in addition to the chain-melting transition, in both the heating and cooling modes. The gel states of these even-numbered compounds are characterized by a relatively greater mobility of the phosphate head group as seen by 31P NMR spectroscopy. The differences between the odd-numbered and even-numbered compounds are reflected in a pronounced odd-even alternation in the characteristic transition temperatures and enthalpies and in differences in their responses to changes in the composition of the bulk aqueous phase. Moreover, both the odd-numbered and even-numbered omega-cyclohexylphosphatidylcholines exhibit significantly lower chain-melting transition temperatures and enthalpies than do linear saturated phosphatidylcholines of comparable chain length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential scanning calorimetry studies of some analogs for the lipid component of biological membranes

The melting behavior of members of newly synthesized series of rac-1,2-diglycerides with substituted phenyl groups or a benzyl group on the 3-position was investigated with differential scanning calorimetry (DSC). Solution crystallized samples had single melting temperatures, higher than those of the quenched or annealed specimens. Quenched samples exhibited polymorphic behavior; some melted and recrystallized during slow heating. This behavior is similar to that of lecithins and suggests that X-ray diffraction studies of the substituted diglycerides may be useful for understanding membrane structure and functions.     

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.     

Differential scanning calorimetric studies of the thermotropic phase behavior of membranes composed of dipalmitoyllecithin and mixed-acid unsaturated lecithins

The lecithins 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) have been synthesized by reacylation of the appropriate lysolecithins with fatty acid anhydrides. These lecithins have been used to make model membranes in mixtures with dipalmitoyllecithin (DPPC), and phase diagrams of the two bilayer systems have been constructed. These diagrams show that there is essentially no gel-state miscibility in the POPC-DPPC bilayers at any composition, and that SOPC-DPPC bilayers show gel-state immiscibility at DPPC concentrations of less than 50 mol%, and partial miscibility above 50 mol% DPPC. Analysis of the POPC-DPPC phase diagram on the assumption of athermal solution in the liquid-crystalline phase shows that the two lipids mix nearly randomly above the phase transition. The liquidus curve of SOPC-DPPC bilayers showed deviations from calculated ideal behaviour, which indicated that there is a small excess tendency for the formation of pairs of like molecules in SOPC-DPPC bilayers in the liquid-crystalline phase. Thus, in the liquid-crystalline phase, SOPC and DPPC do not pack quite as well as do POPC and DPPC.     

Differential scanning calorimetry studies of the inverse temperature transition of the polypentapeptide of elastin and its analogues

Differential scanning calorimetry studies have been carried out on the sequential polypeptide of elastin, (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n, abbreviated as PPP, and its more hydrophobic analogues (L-Leu1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Leu1-PPP, and (L-Ile1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Ile1-PPP Consistent with inverse temperature transitions, the temperatures of the transitions for which maximum heat absorption occurs are inversely proportional to the hydrophobicities of the polypentapeptides (31 degrees C for PPP, 16 degrees C for Leu1-PPP, and 12 degrees C for Ile1-PPP), and the endothermic heats of the transitions are small and increase with increasing hydrophobicity, i.e., 1.2, 2.9, and 3.0 kcal/mol pentamer for PPP, Leu1-PPP, and Ile1-PPP, respectively. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order, as the temperature is raised above that of the transition, has been repeatedly observed using different physical characterizations. Furthermore, the studies demonstrated identical conformations for PPP and Il21-PPP above and below the transition. Both heats and temperatures of the transitions vary with hydrophobicity, but not in simple proportionality.     

Differential scanning calorimetry and X-ray diffraction studies of a series of synthetic beta-D-galactosyl diacylglycerols.

We have investigated the physical properties of a homologous series of synthetic, saturated 1,2-di-O-acyl-3-O-(beta-D-galactopyranosyl)-sn-glycerols using calorimetry and X-ray diffraction. Unannealed aqueous dispersions of these compounds exhibit a lower temperature, moderately energetic, chain-melting (L beta/L alpha) phase transition and a higher temperature, weakly energetic, bilayer/nonbilayer phase transition. On annealing below the L beta/L alpha phase transition, the L beta phase converts to an LC phase, which may undergo a highly energetic LC/L alpha or LC/HII phase transition at very high temperatures on reheating. The temperatures of these phase transitions are higher than those seen in the corresponding alpha- and beta-D-glucosyl diacylglycerols. However, the L beta/L alpha and bilayer/nonbilayer phase transition temperatures of the beta-D-galactosyl diacylglycerols are lower than those of the corresponding diacyl phosphatidylethanolamines. These observations are discussed in terms of the hydration and hydrogen bonding properties of their respective headgroups.     

Differential scanning calorimetry study of glycogen phosphorylaseb-detergent interactions

The overall thermal denaturation of glycogen phosphorylaseb is irreversible and our results conform to the theoretical prediction of a reversible process followed by a slower irreversible process. The basic thermodynamic parameters of glycogen phosphorylaseb denaturation have been worked out and found to be: critical temperature 57.0±0.5°C, transition half-width 8±1°C, and calorimetric enthalpy change and Van't Hoff enthalpy change of the denaturation process 450±50 and 105±15 kcal/mol of enzyme monomer, respectively, at pH 7.4. These parameters have been found to be largely altered by the detergents octylglucoside, cholate, and deoxycholate at or below their critical micelle concentration, but not by Triton X-100 nor by lecithin liposomes. Organic solvents, such as dimethyl sulfoxide and methanol, and the presence of sarcoplasmic reticulum membranes produces an alteration of the denaturation thermogram of glycogen phosphorylaseb similar to that produced by the above-mentioned detergents. These results allow us to hypothesize that hydrophobic domains of glycogen phosphorylaseb are involved in its association to sarcoplasmic reticulum membranes in the sarcoplasmic reticulum/glycogenolytic complex of mammalian skeletal muscle.     

Differential scanning calorimetry of antitumor antibiotics-plasmid DNA interaction

Differential scanning calorimetry (DSC) can detect stepwise melting of plasmid DNA along the molecular chain with high resolution. This method was applied to study interaction of some antitumor antibiotics with the plasmid pJL3-TB5 DNA (5277 base-pairs in length). Analysis of DSC curves of the plasmid DNA in the presence of, for example, adriamycin, an antitumor antibiotics of anthracycline group, together with theoretical analysis of the DNA melting curves obtained by calculation from the entire base sequence, led to the conclusion that adriamycin bound preferentially to the four particular regions with high G + C content. The DSC method would thus be useful for the study of properties of drugs which bind to DNA.     

Differential scanning calorimetry of chicken erythrocyte nuclei.

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98 degrees C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher-order structure is stabilized by fibronectin-like proteins.     

Differential scanning calorimetry of milk fat globule membranes

Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned.     

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.     

Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin

A differential scanning calorimetry study of the thermal denaturation of Bacillus thermoproteolyticus rokko thermolysin was carried out. The calorimetric traces were found to be irreversible and highly scan-rate dependent. The shape of the thermograms, as well as their scan-rate dependence, can be explained by assuming that the thermal denaturation takes place according to the kinetic scheme N k----D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation, N the native state, and D the unfolded state or, more probably, a final state, irreversibly arrived at from the unfolded one. On the basis of this model, the value of the rate constant as a function of temperature and the activation energy have been calculated. It is shown that the proposed model may be considered as being one particular case of that proposed by Lumry and Eyring [Lumry, R., & Eyring, H. (1954) J. Phys. Chem. 58, 110] N in equilibrium D----I, where N is the native state, D the unfolded one, and I a final state, irreversibly arrived at from D. Lastly, some comments are made on the use of the scan-rate effect on the calorimetric traces as an equilibrium criterion in differential scanning calorimetry.