A differential scanning calorimetry study of tetracycline repressor.

Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix-turn-helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: N(2)--->D(2), where k is a first-order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two-state model: N(2)<--->U(2)--->D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DeltaH(cal) = 1067 kJ x mol(-1)) and an entropy change (DeltaS = 3.1 kJ x mol(-1)).     

Binding study of riboflavin-binding protein with riboflavin and its analogues by differential scanning calorimetry

Thermal unfolding parameters of hens' egg-white riboflavin-binding-protein (RBP) were measured by differential scanning calorimetry. Thermal denaturation scans of apoRBP and RBP complexes with riboflavin and its analogues (FMN, N10 DL-glyceryl isoalloxazine, and N10 -hydroxypentyl isoalloxazine) have been measured. It was found that ligand binding causes increase of RBP thermal stability, as manifested by a change of denaturation temperature from 60.8°C for apoRBP to 72.8°C for RBP—Rf complex. For RBP—FMN complex, the denaturation temperature of 73.0°C was even higher than for the RBP—Rf complex. The other two flavin analogues showed transition temperatures in between 66.9°C and 68.8°C, respectively. Analysis of excess heat capacity data showed that the best fit was the sum of two independent thermal transitions. One of the transitions, which contributed 70% to the total heat effect, has transition temperature in the broad range of 60.5–73.2°C; the other transition temperature is in the narrower range of 65.4–71.1°C. The observed transitions can be related to RBP domains.     

A differential scanning calorimetry study of the interaction of alpha-tocopherol with mixtures of phospholipids

When alpha-tocopherol was included in multibilayer vesicles of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine it induced a broadening of the main transition and a displacement of this transition to lower temperatures, as seen by differential scanning calorimetry. This effect was quantitatively more important in the samples of distearoylphosphatidylcholine than in those of the other phosphatidylcholines. Alpha-Tocopherol when present in equimolar mixtures of dimyristoylphosphatidylcholine and diastearoylphosphatidylcholine, which show monotectic behaviour, preferentially partitions in the most fluid phase. The effect of alpha-tocopherol on the phase transition of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine is qualitatively different of that observed on phosphatidylcholines, and several peaks are observed in the calorimetric profile, probably indicating the formation of separated phases with different contents in alpha-tocopherol. The effect was more apparent in dipalmitoylphosphatidylethanolamine than in dilauroylphosphatidylethanolamine. The inclusion of alpha-tocopherol in equimolar mixtures of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylcholine, which show cocrystallization, only produced a broadening of the phase transition and a shift to lower temperatures. However, in the case of equimolar mixtures of dipalmitoylphosphatidylcholine which also show cocrystallization, the effect was to cause lateral phase separation with the formation of different mixtures of phospholipids and alpha-tocopherol. Alpha-Tocopherol was also included in equimolar mixtures of phosphatidylethanolamine and phosphatidylcholine showing monotectic behaviour, and in this case alpha-tocopherol preferentially partitioned in the most fluid phase, independently of whether this was composed mainly of phosphatidylcholine or of phosphatidylethanolamine.     

A differential scanning calorimetry study of the interaction of lasalocid antibiotic with phospholipid bilayers

Interaction of lasalocid sodium salt (Las-Na) with dipalmitoylphosphatidylcholine (DPPC) as a membrane model was investigated by highly-sensitive differential scanning calorimetry (DSC). The insertion properties of the antibiotic were studied both in multilamellar suspensions and unilamellar vesicles, for Las-Na/DPPC molar ratios (r) ranging from 0.005 to 0.1. The effect of the antibiotic on the lipid thermotropic behavior is concentration dependent and drastically changes at a critical r of 0.04 in both model membranes. Below this ratio, Las-Na molecules interact with DPPC bilayers without disrupting the global organization of the membrane. In the multilamellar systems only the transition cooperativity is affected whereas for the mixed vesicles, a decrease in the enthalpy change suggests a different mode of insertion. Above this ratio, implantation of the antibiotic give rise to lateral phase separation in multilamellar systems. These structural modifications have repercussions on the formation of mixed LAS-Na/DPPC vesicles which seems limited to an r value of 0.04.     

A differential scanning calorimetry study of acetylcholine receptor-rich membranes from Torpedo californica

Various acetylcholine receptor-rich membrane preparations from Torpedo californica electroplax tissue were examined using the techniques of differential scanning calorimetry coupled with gel electrophoretic analysis of heat-denaturing material and functional assays following passage through discrete transitions. In unfractionated membranes, four irreversible calorimetric transitions were observed, one of which (Td = 59 degrees C) could be assigned to a complete loss of acetylcholine receptor function. A second lower temperature transition apparently corresponds to loss of certain peripheral membrane proteins including the Mr = 43,000 polypeptide and the acetylcholinesterase activity. Membrane preparations highly enriched in acetylcholine receptor polypeptides contained a major transition at 59 degrees C which could be shown to be sensitive to the presence of added ligands of the acetylcholine receptor, supporting its assignment to structural alterations of the receptor protein or its arrangement in the membrane.     

Multidomain structure of a recombinant streptokinase. A differential scanning calorimetry study

The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.     

A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes

Mixtures of stearic, arachic, oleic and linoleic acids with dimyristoylphosphatidylcholine (DMPC) and distearylphosphatidylcholine (DSPC) have been studied by means of differential scanning calorimetry (DSC). The mixtures of stearic (SA) and arachic acids (AA) with DMPC and DSPC show phase diagrams of the peritectic type, with a region of solid phase immiscibility from 0 to 28.5 mol% of fatty acid. A pure component, with a stoichiometry fatty acid/phospholipid (2:1) seems to be formed except for the system AA/DSPC. The mixtures of oleic (OA) and linoleic acids (LA) show complex phase diagrams. In the case of OA, different regions where a phase separation exists can be observed and for the mixture of OA with DMPC, a pure component seems to be formed with a stoichiometry OA/DMPC (1:2). LA shows different behaviour in the mixtures with DMPC and with DSPC. For the mixture, LA/DMPC, a fluid phase immiscibility region is observed over the same range of concentration as for the mixture with OA, however, the mixture with DSPC shows a solid phase immiscibility for the samples containing 45 mol% or more of LA. The interaction of the different free fatty acids with equimolar mixtures of DMPC and DSPC, showing monotectic behaviour, has also been analyzed. From our results it can be clearly concluded that saturated fatty acids partition preferentially into solid-like domains, while cis-unsaturated fatty acids partition preferentially into fluid-like domains.     

Interaction of retinol and retinoic acid with phospholipid membranes. A differential scanning calorimetry study.

The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer.     

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.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

A differential scanning calorimetry study of the interaction of gangliosides with peanut lectin,serotonin and daunomycin

Thermotropic behaviour of human Tay-Sachs ganglioside and of mixed bovine brain gangliosides, before and after interaction with peanut lectin, serotonin and daunomycin, was investigated. Interaction of mixed brain gangliosides with peanut lectin or serotonin causes a decrease in the enthalpy of melting, whereas interaction of this lectin with Tay-Sachs ganglioside does not influence the enthalpy of melting. Serotonin causes a small increase in the enthalpy of melting of the Tay-Sachs ganglioside.     

Dantrolene inhibits halothane-induced membrane reorganization A study using 31P-NMR and differential scanning calorimetry

The action of the relaxing agent dantrolene on dipalmitoylphosphatidylcholine (DPPC) model membranes in the presence and absence of the general anesthetic halothane has been investigated by DSC and 31P-NMR. Dantrolene has a weak effect on both the thermodynamic and NMR parameters of the pure model membrane. When halothane is present in the system, the relaxing agent acts to counterbalance the strong anesthetic-induced membrane perturbation. This is reflected in DSC experiments by a change of the enthalpy variation (delta H) and of the main gel-to-fluid phase transition temperature (Tc) towards the values of the pure lipid system. The amount of halothane-induced small tumbling vesicles, as detected by 31P-NMR by the superposition of an isotropic line on a lamellar-type powder spectrum, is considerably reduced upon dantrolene addition. This means that the relaxing agent "cures" the membrane de-structuring action promoted by halothane. Membranes first treated with dantrolene are also protected from the halothane perturbation. So, the relaxing agent is both "curative" and "preventative" against halothane. The optimum effect is obtained for 1 dantrolene molecule per ca 34 halothane molecules. The mechanisms of action were discussed in relation to membrane fluidity.     

Dynamic analysis of differential scanning calorimetry data

The apparent heat capacity function measured by high-sensitivity differential scanning calorimetry contains dynamic components of two different origins: (1) an intrinsic component arising from the finite instrument time response; and (2) a sample component arising from the kinetics of the thermal transition under study. The intrinsic instrumental component is always present and its effect on the shape of the experimental curve depends on the magnitude of the calorimeter response time. Usually, high-sensitivity instruments exhibit characteristic time constants varying from 10 to 100 s. This slow response introduces distortions in the shape of the heat capacity function especially at fast scanning rates. In addition to this instrumental component, dynamic effects due to sample relaxation processes also contribute to the shape of the experimental heat capacity profile. Since the nature and magnitude of these effects are a function of the kinetic parameters of the transition, they can be used to obtain kinetic information. This communication presents a dynamic deconvolution technique directed to remove artificial distortions in the shape of the heat capacity function measured at any scanning rate, and to obtain a kinetic characterization of a thermally induced transition. The kinetic characterization obtained by this method allows the researcher to obtain transition relaxation times as a continuous function of temperature. This technique has been applied to the thermal unfolding of ribonuclease A and the pretransition of dipalmitoylphosphatidylcholine (DPPC). In both systems the transition relaxation times are temperature dependent. For the protein system the relaxation time is very slow below the transition temperature (approximately 30 s) and very fast above Tm (less than 1 s) in agreement with direct kinetic measurements. For the pretransition of DPPC, the relaxation time is maximal at the transition midpoint and of the order of approx. 40 s.     

Interactions of glycerol monooleate and dimethylsulphoxide with phospholipids A differential scanning calorimetry and 31P-NMR study

1. A comparative study has been made of the effects of the fusogens glycerol monooleate and dimethylsulphoxide on the polymorphic phase behaviour of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine by differential scanning calorimetry and ³¹P-NMR techniques. 2. Glycerol monooleate induces a reduction in the temperature, cooperativity and enthalpy of the gel to liquid-crystal transitions of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine, whereas dimethylsulphoxide induces an increase in the temperature and enthalpy and a reduction in the cooperativity of the gel to liquid-crystal transitions for those same phospholipids. 3. Glycerol monooleate induces the formation of isotropic and hexagonal (H_II) phases when mixed with either dipalmitoyl phosphatidylcholine or dipalmitoyl phosphatidylethanolamine. By contrast, in the presence of dimethylsulphoxide, those same phospholipids retain the lamellar configuration observed in the absence of fusogen. 4. These results are discussed in terms of the mechanisms of chemically induced cell fusion.     

Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study

Phaseolus vulgaris phytohemagglutinin L is a homotetrameric-leucoagglutinating seed lectin. Its three-dimensional structure shows similarity with other members of the legume lectin family. The tetrameric form of this lectin is pH dependent. Gel filtration results showed that the protein exists in its dimeric state at pH 2.5 and as a tetramer at pH 7.2. Contrary to earlier reports on legume lectins that possess canonical dimers, thermal denaturation studies show that the refolding of phytohemagglutinin L at neutral pH is irreversible. Differential scanning calorimetry (DSC) was used to study the denaturation of this lectin as a function of pH that ranged from 2.0 to 3.0. The lectin was found to be extremely thermostable with a transition temperature around 82 degrees C and above 100 degrees C at pH 2.5 and 7.2, respectively. The ratio of calorimetric to vant Hoff enthalpy could not be calculated because of its irreversible-folding behavior. However, from the DSC data, it was discovered that the protein remains in its compact-folded state, even at pH 2.3, with the onset of denaturation occurring at 60 degrees C.     

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scanning calorimetry

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. From this model, the activation energies obtained for the denaturation of the pro- and carboxypeptidase were 300 +/- 20 kJ mol-1 and 250 +/- 14 kJ mol-1 respectively. On the other hand, the isolated activation segment appears as a thermostable piece with a highly reversible thermal unfolding which follows a two-state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the active enzyme.     

Study of the thermal behavior of azidohetarenes with differential scanning calorimetry

Thermal properties of azidohetarenes without reactive ortho-substituents, obtained by DSC analysis, were compared with the DSC data of ortho-phenyl and ortho-acyl substituted azidohetarenes, which give ring closure reactions either to indoles or to five-membered heterocycles such as isoxazoles. The reaction temperatures and reaction enthalpies give both information to prepare the reaction conditions and important safety information and were, in addition, used to find out relations between the temperatures or enthalpies with the three different reaction mechanisms.     

Physical-chemical characterization and stability study of alpha-trypsin at pH 3.0 by differential scanning calorimetry

alpha-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel ss-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of alpha-trypsin in the acid pH range was performed and some physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The alpha-trypsin has a shelf-life (t(95%)) of about 10 months at pH 3.0 and 4 degrees C and its hydrolysis into the psi-trypsin isoform is negligible during 6 months. The observed ratio DeltaH(cal)/DeltaH(vH) is close to unity, which suggests the occurrence of a two-state transition. At pH 3.0, alpha-trypsin unfolded with T(m) = 325.9 K and DeltaH = 99.10 kcal mol(-1), and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96+/-0.18 kcal mol(-1)K(-1). The stability of alpha-trypsin calculated at 298 K was DeltaG(U)=6.10 kcal mol(-1) at pH 3.0. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters of unfolding of beta-trypsin do not change substantially after its conversion to alpha-trypsin.