Thermotropic and mixing behavior of mixed-chain phosphatidylcholines with molecular weights identical with that of L-alpha-dipalmitoylphosphatidylcholine.

The thermotropic phase behavior of 10 mixed-chain phosphatidylcholines, in excess water, has been examined and compared with that of identical-chain C(16):C(16)PC by using high-resolution differential scanning calorimetry (DSC). The molecular weights (MW) of these 11 molecular species are the same, but their delta C/CL values, or the normalized chain length differences, vary considerably, ranging from 0.035 to 0.540. The thermodynamic parameters (Tm, delta H, and delta S) associated with the main phase transitions for these lipid dispersions exhibit biphasic V-shaped curves, when plotted against delta C/CL. Similar characteristic curves have been reported previously for aqueous dispersions of mixed-chain phosphatidylcholines with MW identical with that of C(17):C(17)PC [Lin et al. (1990) Biochemistry 29, 7063-7072]. The initial decrease in Tm (delta H or delta S) with increasing values of delta C/CL is attributed to the progressive increase in the magnitude of the chain-terminal perturbations on the conformational statistics of the adjacent hydrocarbon chains and hence the lateral chain-chain interactions of these mixed-chain phosphatidylcholines in the gel-state bilayer. At delta C/CL approximately equal to 0.42, the chain-end perturbation is presumably at its maximum; beyond this point, the highly asymmetric phosphatidylcholines are proposed to pack, at T less than Tm, into the mixed interdigitated bilayer. In this new packing mode, the methyl ends of the longer acyl chains are relocated at the interfaces between the hydrocarbon core of the bilayer and the aqueous medium. This disposition of the bulky chain ends releases a certain degree of chain-chain packing disorders, leading to an increase in Tm (delta H or delta S) with increasing delta C/CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binary mixtures of saturated and unsaturated mixed-chain phosphatidylcholine. A differential scanning calorimetry study

High-resolution differential scanning calorimetry (DSC) has been used to study the aqueous dispersions of mixed-chain phosphatidylcholines prepared from colyophilized mixtures of C(18):C(11:1 delta 10) PC/C(18):C(10)PC and C(18):C(11:1 delta 10) PC/C(18):C(11)PC of various molar ratios. These mixed-chain phospholipids are characterized by a marked disparity in their acyl-chain lengths; however, the sn-1 acyl chain in the fully extended conformation is about twice as long as the sn-2 acyl chain. Their thermotropic behavior was determined, and the phase diagrams of these two mixtures were constructed from the calorimetric data. Results indicate that C(18):C(11:1 delta 10)PC/C(18):C(10)PC and C(18):C-(11:1 delta 10)PC/C(18):C(11)PC are miscible in all proportions with a near-ideal behavior of mixing in the gel and liquid-crystalline phases. Equimolar mixtures of diC(14)PC/C(18):C(11:1 delta 10)PC, diC(14)PC/C(18):C(10)PC, and diC(14)PC/C(18):C(11)PC have also been studied by DSC. These phosphatidylcholines in the 1:1 mixture differ in Tm by less than 11 degrees C; however, they exhibit gel-phase immiscibility in the plane of the bilayer. Taken together, these studies suggest that C(18):C(11)PC and C(18):C(11:1 delta 10)PC are packed similarly to C(18):C(10)PC in excess water as mixed interdigitated bilayers, at T less than Tm, which transform into partially interdigitated bilayers when heated above Tm.     

Mixing behavior of identical molecular weight phosphatidylcholines with various chain-length differences in two-component lamellae.

It has recently been suggested that mixed-chain phosphatidylcholines with normalized chain length differences (deltaC/CL) in the range of 0.10-0.40 undergo spontaneous self-assembly in excess water at T less than Tm into the partially interdigitated bilayer and those with delta C/CL values in the range of 0.44-0.57 form, in excess water, mixed interdigitated bilayers at T less than Tm. The mixing behavior of binary mixtures of C(22):C(12)PC/C(17):C(17)PC, C(22):C(12)/C(15):C(19)PC, and C(15):C(19)PC/C(13):C(21)PC reported in this work is used to support this view. The values of delta C/CL for C(17):C(17)PC, C(15):C(19)PC, C(13):C(21)PC, and C(22):C(12)PC are 0.10, 0.15, 0.35, and 0.55, respectively. The binary mixture of C(15):C(19)PC/C(13):C(21)PC exhibits a lens-shaped phase diagram, indicating that these two identical molecular weight (MW) lipids with delta C/CL values less than 0.4 are completely miscible over the entire compositional range in both gel and liquid-crystalline phases. In contrast, the phase diagrams of C(22):C(12)PC/C(17):C(17)PC and C(22):C(12)PC/C(15):C(19)PC are eutectic, indicating immiscibility of the component lipids over a wide compositional range in the gel phase. This immiscibility of identical MW lipids in the bilayer plane can be attributed to the different packing properties of the component lipids in the bilayer at T less than Tm.     

Differential scanning calorimetric study of a homologous series of fully hydrated saturated mixed-chain C(X):C(X + 6) phosphatidylcholines

The successive high-resolution differential scanning calorimetric (DSC) thermograms for aqueous dispersions of a homologous series of mixed-chain phosphatidylcholines, C(X):C(X + 6)PC, have been recorded and analyzed. In this series of saturated mixed-chain phosphatidylcholines, the total number of carbon atoms in the sn-1 acyl chain increases from 11 to 20, and the sn-2 acyl chain is always 6 methylene units longer than the sn-1 acyl chain. In the initial heating DSC thermograms, two prominent endothermic transitions are detected for all the samples prepared from the various C(X):C(X + 6)PCs except C(12):C(18)PC. In contrast, a single exothermic transition is observed on cooling for all the samples except C(13):C(19)PC. The temperature difference between the two endothermic transitions increases linearly as the acyl chain length of C(X):C(X + 6)PC becomes progressively longer. Interestingly, the main phase transition occurs before the subtransition for C(11):C(17)PC dispersions. Our DSC data further demonstrate that the thermodynamic parameters (Tm, delta H, and delta S) associated with the main phase transition for fully hydrated C(13):C(19)PC and other identical MW phosphatidylcholines are inversely related to the corresponding values of the chain-length inequivalence (delta C/CL) for these lipids. This linear relationship can be employed to map the Tm values for aqueous dispersions prepared from a large number of mixed-chain phosphatidylcholines whose values of delta C/CL are within the range of 0.1-0.4.     

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.     

Energy-minimized structures and packing states of a homologous series of mixed-chain phosphatidylcholines: a molecular mechanics study on the diglyceride moieties.

Phosphatidylcholines or C(X):C(Y)PC, quantitatively the most abundant lipids in animal cell membranes, are structurally composed of two parts: a headgroup and a diglyceride. The diglyceride moiety consists of the glycerol backbone and two acyl chains. It is the wide diversity of the acyl chains, or the large variations in X and Y in C(X):C(Y)PC, that makes the family of phosphatidylcholines an extremely complex mixture of different molecular species. Since most of the physical properties of phospholipids with the same headgroup depend strongly on the structures of the lipid acyl chains, the energy-minimized structure and steric energy of each diglyceride moiety of a series of 14 molecular species of phosphatidylcholines with molecular weights identical to that of dimyristoylphosphatidylcholine without the headgroup are determined in this communication by molecular mechanics (MM) calculations. Results of two types of trans-bilayer dimer for each of the 14 molecular species of phosphatidylcholines are also presented; specifically, the dimeric structures are constructed initially based on the partially interdigitated and mixed interdigitated packing motifs followed subsequently by the energy-minimized refinement with MM calculations. Finally, tetramers with various structures to model the lateral lipid-lipid interactions in a lipid bilayer are considered. Results of laborious MM calculations show that saturated diacyl C(X):C(Y)PC with delta C/CL values greater than 0.41 prefer topologically to assemble into tetramers of the mixed interdigitated motif, and those with delta C/CL values less than 0.41 prefer to assemble into tetramers with a repertoire of the partially interdigitated motif. Here, delta C/CL, a lipid asymmetry parameter, is defined as the normalized acyl chain length difference between the sn-1 and sn-2 acyl chains for a C(X):C(Y)PC molecule; an increase in delta C/CL value is an indication of increasing asymmetry between the two lipid acyl chains. These computational results are in complete accord with the calorimetric data presented previously from this laboratory (H-n. Lin, Z-q. Wang, and C. Huang. 1991. Biochim. Biophys. Acta. 1067:17-28).     

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.     

Differential scanning calorimetry of a conformational transition in heavy meromyosin

We have investigated the potential use of differential scanning calorimetry (DSC) to characterize conformational changes in proteins with emphasis on a conformational change in the myosin head which may be related to the power-stroke providing force production in muscle contraction. Simulations indicate that two-state conformational transitions with enthalpy changes greater than approximately 30 kcal/mol should be observable by DSC. We present here differential scanning calorimetric studies of a predenaturation structural change in heavy meromyosin. The high concentration of protein required for these experiments leads to potential contributions from intermolecular interactions. The technical difficulties associated with studying conformational transitions by DSC are discussed.     

Differential scanning calorimetry and X-ray diffraction study of tendon collagen thermal denaturation

Differential scanning calorimetry, high and small angle X-ray diffraction analyses have been carried out on air-dried and rehydrated rat tail tendon collagen in order to test the reversibility of collagen thermal denaturation. The mean enthalpy values calculated for the denaturation process of air-dried and rehydrated samples are ΔH_D = 9.0 ± 0.8 cal/g and ΔH_D = 11.9 ±0.7 cal/g respectively, while the denaturation temperatures are T_D = 112 ± 1°C and T_D = 51 ± 1°C. Partial reversibility of the coiled coil—random coil process can be obtained by storing the samples in air or more rapidly by equilibration in water. After denaturation air-dried collagen fibres recover not only their molecular structure but also their characteristic fibrillar structure. The latter does not greatly influence the mean experimental enthalpy values.     

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 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.     

Probing the ethanol-induced chain interdigitations in gel-state bilayers of mixed-chain phosphatidylcholines

Using high-resolution differential scanning calorimetry (DSC), we have studied the effects of ethanol concentrations, [EtOH], on the main phase transition temperatures (T[m]) of the following mixed-chain phosphatidylcholines (PCs): C(15):C(17)PC, C(17):C(15)PC, and C(12):C(20)PC. These lipids have a common molecular weight; however, their apparent acyl chain-length differences between the sn-1 and sn-2 acyl chains, delta C, are distinctively different. The delta C values for these three mixed-chain PCs are, respectively, 0.5, 3.5, and 6.5 C-C bond lengths. DSC results show that the T(m) profiles for C(15):C(17)PC and C(17):C(15)PC bilayers in the plot of T(m) versus [EtOH] are V-shaped biphasic curves, with the minimum T(m) occurring at 50 and 73 mg/ml of ethanol, respectively. In contrast, the C(12):C(20)PC bilayer exhibits a nearly linear decrease in T(m) with increasing [EtOH]. In addition, x-ray diffraction experiments were also performed to assess the structural changes of these three mixed-chain PCs in the gel-state bilayers, at 20 degrees C, in response to high concentrations of ethanol. X-ray diffraction data indicate that, in the absence of ethanol, these three lamellar lipids are all packed in the normal (L beta') gel phase in aqueous media. In the presence of 120 mg/ml of ethanol, however, the C(15):C(17)PC and C(17):C(15)PC lamellae are packed in the fully interdigitated (L beta[I]) gel phase. The V-shaped T(m) curves detected calorimetrically for these two lipids in response to [EtOH] can thus be explained by the ethanol-induced L beta' --> L beta[I] isothermal phase transition. Interestingly, the results of x-ray diffraction study reveal, for the first time, that an ethanol-induced L beta' --> L(MI) (mixed interdigitated phase) isothermal phase transition occurs in the gel-state bilayer of highly asymmetrical C(12):C(20)PC. Therefore, the chain asymmetry is recognized to play an important role in the ethanol-induced chain interdigitation at T < T(m).     

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 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.     

Effects of alcohols on the phase transition temperatures of mixed-chain phosphatidylcholines.

The biphasic effect of ethanol on the main phase transition temperature (Tm) of identical-chain phosphatidyl-cholines (PCs) in excess H2O is now well known. This biphasic effect can be attributed to the transformation of the lipid bilayer, induced by high concentrations of ethanol, from the partially interdigitated L beta, phase to the fully interdigitated L beta I phase at T < Tm. The basic packing unit of the L beta I phase has been identified recently as a binary mixture of PC/ethanol at the molar ratio of 1:2. The ethanol effect on mixed-chain PCs, however, is not known. We have thus in this study investigated the alcohol effects on the Tm of mixed-chain PCs with different delta C values, where delta C is the effective acyl chain length difference between the sn-1 and sn-2 acyl chains. Initially, molecular mechanics (MM) simulations are employed to calculate the steric energies associated with a homologous series of mixed-chain PCs packed in the partially and the fully interdigitated L beta I motifs. Based on the energetics, the preference of each mixed-chain PC for packing between these two different motifs can be estimated. Guided by MM results, high-resolution differential scanning calorimetry is subsequently employed to determine the Tm values for aqueous lipid dispersions prepared individually from a series of mixed-chain PCs (delta C = 0.5-6.5 C-C bond lengths) in the presence of various concentrations of ethanol. Results indicate that aqueous dispersions prepared from mixed-chain PCs with a delta C value of less than 4 exhibit a biphasic profile in the plot of Tm versus ethanol concentration. In contrast, highly asymmetric PCs (delta C > 4) do not exhibit such biphasic behavior. In the presence of a longer chain n-alcohol, however, aqueous dispersions of highly asymmetric C(12):C(20)PC (delta C = 6.5) do show such biphasic behavior against ethanol. Our results suggest that the delta C region in a highly asymmetric PC packed in the L beta I phase is most likely the binding site for n-alcohol.     

Differential scanning calorimetry characterization of oxidized egg phosphatidylcholine liposomes

Differential scanning calorimetry has been used to characterize liposomes made from mixtures of unoxidized and singlet oxygen oxidized egg phosphatidylcholine. Cooling scans reveal that trapped water decreases when the oxidized phosphatidylcholine content is increased in the liposomes. Liposomes made from mixtures containing more than 50% by weight of oxidized phosphatidylcholine do not show trapped water.     

Cholesterol effects on a mixed-chain phosphatidylcholine bilayer: a molecular dynamics simulation study

A molecular dynamics simulation of a mono-cis-unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer containing approximately 22 mol% of cholesterol (POPC-Chol) was carried out for 15 ns. An 8-ns trajectory was analysed to determine the effects of Chol on the membrane properties and compare it with that on the fully saturated 1,2-dimyristoyl-phosphatidylcholine bilayer containing approximately 22 mol% of Chol (DMPC-Chol). The study suggests that the experimentally observed weaker effect of Chol on the POPC than DMPC bilayer might result from a different vertical localisation of the Chol hydroxyl group (OH-Chol) in both bilayers: in the POPC-Chol bilayer, OH-Chol is placed approximately 3 A higher in the bilayer interface than in the DMPC-Chol bilayer. Because of the rigid cis double bond in the beta-chain of POPC, Chol fits worse to the POPC-Chol membrane environment and is pushed up, in effect all Chol ring atoms are, on average, located above the double bond. Both in mono-cis-unsaturated and fully saturated PC bilayers, Chol induces stronger van der Waals interactions among the chains, whereas its interactions with the chains are weak. In contrast to DMPC, the smooth alpha-face of the Chol ring lowers the order of POPC chains, whereas the rough beta-face increases the order.     

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.     

Differential scanning calorimetry of chromatin at different levels of condensation

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 °C, while the other two occur at 63 °C and 74 °C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 °C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.