Thermal phase transitions in sheep, rat and rabbit surfactant lipids detected by differential scanning calorimetry

1. The thermal behaviour of sheep, rat and rabbit pulmonary surfactant lipids was investigated using high sensitivity differential scanning calorimetry (DSC). 2. Phase transitions were evident in the surfactant lipids from all three animals, with the upper limit of the phase transition being 30.1 C in the sheep, 36.8 C in the rat and 36.3 C in the rabbit. 3. The relatively greater fluidity of the sheep surfactant lipids in comparison to those of the rat and rabbit was due primarily to differences in their palmitic acid content.     

Reversible fast-dimerization of bovine serum albumin detected by fluorescence resonance energy transfer

Self-association of bovine serum albumin (BSA) was explored using fluorescence resonance energy transfer (FRET) between two populations of the protein labeled separately with either fluorescein-5'-isothiocyanate (FITC) or eosin-5'-isothiocyanate (EITC). The energy transfer reached the steady state after 5 s at 25 degrees C, indicating a fast exchange between oligomer subunits. The dependence of the energy transfer efficiency on the protein concentration and its reversion by unlabeled BSA demonstrate that association between BSA monomers occurs through a reversible path that involves specific interactions between the protein molecules. Because energy transfer took place even after blocking Cys 34 with iodoacetamide, this residue might not be involved in the reversible self-association process. The number of subunits forming the oligomer and its dissociation constant were determined from measurements of energy transfer as a function of the donor-acceptor ratio and of the total protein concentration. Analysis of these data indicated that BSA is in a monomer-dimer equilibrium with a dissociation constant of 10 +/- 2 microM at 25 degrees C in 10 mM MOPS-K (pH 5.8).     

Low-temperature glass transitions of quenched and annealed bovine serum albumin aqueous solutions

To investigate the glass transition behaviors of a 20% (w/w) aqueous solution of bovine serum albumin, heat capacities and enthalpy relaxation rates were measured by adiabatic calorimetry at temperatures ranging from 80 to 300 K. One series of measurements was carried out after quenching from 300 down to 80 K and another after annealing in 200-240 K. The quenched sample showed a heat capacity jump indicating a glass transition temperature T(g) = 170 K, and the annealed sample showed a smaller jump with the T(g) shifted toward the higher temperature side. The temperature dependence of the enthalpy relaxation rates for the quenched sample indicated the presence of two enthalpy relaxation effects: one at around 110 K and the other over a wide temperature range (120-190 K). The annealed sample showed three separate relaxation effects giving 1) T(g) = 110 K, 2) 135 K, and 3) temperature higher than 180 K, whereas nothing around 170 K. These effects were thought to originate, respectively, from the rearrangement motions of 1) primary hydrate water forming a direct hydrogen bond with the protein, 2) part of the internal water localized in the opening of a protein structure, and 3) the disordered region in the protein.     

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.     

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.     

Behaviour of radioactive palmitic acid during heat treatment of bovine serum albumin

A radioactive complex AP_2.3 (A: bovine serum albumin, P: radioactive palmitic acid) has been prepared and incubated at pH 9 and 65°C for 60 min. Analysis by disc gel electrophoresis revealed three zones: zone 1, undenatured monomer; zone 1′, modified monomer; and zone 2, dimer. Counting of sliced gels indicated that only zone 1 was radioactive, meaning that fatty acids are released in the process 1→1′, but not in the process 1′+1′→2. In other words, fatty acids are released from albumin when native albumin is unfolded to form component 1′ during the incubation. The fatty acids released are concentrated on a particular species of albumin molecule which is not changed to component 1′. The percentage of component 1 (p) was 37%. This value agrees with that calculated by the equation which was proposed by us in a previous paper, p = (v/6) × 100%. In the present case v is 2.3.     

Kinetics of formation of hydrophobic regions during refolding of bovine serum albumin

The rate of formation of hydrophobic regions during refolding of bovine serum albumin was studied using 1-anilinonaphthalene-8-sulfonate as the hydrophobic fluorescent probe. The refolding of serum albumin exhibited a sigmoidal behavior. The exhibition of a lag phase followed by a faster kinetic phase suggested that the refolding is a cooperative, sequential process. Refolding under reducing conditions almost completely inhibited the regeneration of hydrophobic binding regions, suggesting that the formation of disulfide bonds plays an important role in the refolding of serum albumin. The rate and the extent of refolding was apparently maximum at about 20 degrees; at 37 degrees the extent of refolding was very low compared to that at the other temperatures studied. Based on the results, the mechanism of albumin refolding is interpreted in terms of domain structures and interdomain interactions.     

Raman studies of bovine serum albumin.

The Raman Spectra of bovine serum albumin have been obtained in the solute state, in alkaline and acidic solutions, and in the gel. The reversible denaturations of bovine serum albumin solutions by heat, acid's, and alkali were studied and a new mechanism for heat denaturation has been proposed based on a continuous unfolding of the α-helices.     

Conformational study of human serum albumin in pre-denaturation temperatures by differential scanning calorimetry, circular dichroism and UV spectroscopy

Thermal conformational changes of human serum albumin (HSA) in phosphate buffer, 10 mM at pH = 7 are investigated using differential scanning calorimetric (DSC), circular dichroism (CD) and UV spectroscopic methods. The results indicate that temperature increment from 25 degrees C to 55 degrees C induces reversible conformational changes in the structure of HSA. Conformational change of HSA are shown to be a three-step process. Interestingly, melting temperature of the last domain is equal to the maximum value of fever in pathological conditions, i.e. 42 degrees C. These conformational alterations are accompanied by a mild alteration of secondary structures. Study of HSA-SDS (sodium dodecyl sulphate) interaction at 45 degrees C and 35 degrees C reveals that SDS affects the HSA structure at least in three steps: the first two steps result in more stabilization and compactness of HSA structure, while the last one induces the unfolding of HSA. Since HSA has a more affinity for SDS at 45 degrees C compared to 35 degrees C, It is suggested that the net negative charge of HSA is decreased in fever, which results in the decrease of HSA-associated cations and plasma osmolarity, and consequently, heat removal via the increase in urine volume.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Binding of anandamide to bovine serum albumin

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.     

Interactions of cannabinoids with bovine serum albumin

There is no shift of emission maximum (F₄₇₀nm) of bovine serum albumin (BSA)-l-anilino-8-naphthatene sulphonic acid (ANS) complex in the pesence of delta-9-tetrahydrocannabinol (delta-9-THC) alone and cannabidiol (CBD) or cannabinol (CBN) in the presence and absence of delta-9-THC. Delta-9-THC (1.66–13.33 M) and CBD at higher concentrations (13.33–20.0 M) produce a concentration-dependent significant quenching of fluorescence of BSA-ANS complex, but CBN (l.66–20.0 M) as well as CBD at lower concentrations (1.66–6.66 M) fails to produce any significant change in Iluorescence intensity under similar conditions. Furthermore, neither CBD nor CBN is able to affect the delta-9-THC-induced quenching of fluorescence intensity of BSA-ANS complex. These results indicate that the binding of cannabinoids to the ANS binding sites of BSA molecule are in the order detta-9-THC > CBr3 > CBN, and CBD or CBN does not have any influence on the binding of delta-9-THC to BSA molecules under these conditions.     

Intrinsic viscosity of bovine serum albumin conformers

Intrinsic viscosity of bovine serum albumin (BSA) at different pH values (2.7, 4.3, 7.4, 8 and 10) has been determined, as well as the Mark-Houwink constant and expansion factor. The traditional technique for data analysis using extrapolation to obtain intrinsic viscosity values shows an unusual behavior regarding concentration that can be observed in the values obtained for Huggins' and Kraemer's constants.