Ultrasonic evidence for structural relaxation in large unilamellar liposomes

The ultrasonic absorption of large unilamellar vesicles (average diameter 0.2 micron) was determined in the frequency range 0.5-5 MHz. The liposomes were composed of a 4:1 mixture by weight of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol. They were studied with and without cholesterol or gramicidin incorporated into the bilayer. A large increase in absorption occurs at the solid to liquid-crystalline phase transition temperature (42 degrees C) of the pure lipid vesicles. This increase in absorption is interpreted as a structural relaxation of the 'melting' fatty acid chains occurring with an average relaxation time of 76 ns. The liposomes were also found to be extremely permeable near the transition temperature. Essentially complete release of cytosine arabinoside, a small water-soluble molecule, occurred at 42 degrees C. Addition of cholesterol or gramicidin to the bilayer of the liposomes broadened the ultrasonic absorption and reduced the efflux of cytosine arabinoside at the phase transition. No increase in absorption was observed at the transition temperature in the presence of 50 mol% of cholesterol. Gramicidin, in addition to broadening the transition, slows the isomerization of bonds in the hydrocarbon chains of the lipids. A concentration of 5 mol% gramicidin increased the average relaxation time to 211 ns.     

Helix–coil transition kinetics in aqueous poly(α,L-glutamic acid)

A polarimetric electric-field-jump relaxation apparatus is described and used to determine the relaxation spectrum for the helix–coil transition of poly(α,L -glutamic acid) in water at 24°C. A maximum relaxation time of 1.7 μc occurs at the transition midpoint (pH = 5.9) yielding a rate constant for helical growth of 6 × 10⁷ sec^?1.     

Dielectric Properties of Frozen Biological Material

The origin of the relaxation observed in the organic materials studied here is most likely to be the same as that seen in various frozen water-in-oil emulsions. This conclusion is drawn from the similarity of the activation energies (? 30 kJ/mol), the similar dependences on the ice content and the range of frequencies covered by the relaxation. Observations previously made on the variation of relaxation frequency at ?80°C with time after death of various fish tissues may reflect either a change in the ice fraction of the tissue as protein degradation proceeds resulting in water being released, or water being taken up by the tissue from melting ice during storage at 0°C.     

13C-NMR off-resonance rotating frame spin-lattice relaxation studies of bovine lens γ-crystallin self association: effect of ‘macromolecular crowding’

The NMR technique of ¹³C off-resonance rotating frame spin-lattice relaxation, which provides an accurate assessment of the effective rotational correlation time (τ_0,eff) for macromolecular rotational diffusion, was applied to the study of γ-crystallin association as a function of protein concentration and temperature. Values of the effective rotational correlation time for γ-crystallin rotational diffusion were obtained at moderate to high protein concentrations (80–350 mg/ml) and at temperatures above, and below, the cold cataract phase transition temperature. With increasing concentration γ-crystallin was observed to increasingly associate as reflected by larger values of τ_0,eff Decreasing temperature in the range of 35 to 22°C was found to result in no change in the temperature corrected value of τ_0,eff at a γ-crystallin concentration of 80 mg/ml, whereas at temperatures of 18°C or below, this parameter was approx. twofold larger, suggesting the occurrence of a well defined phase transition, which correlated well with the cold cataract phase transition temperature. At higher protein concentrations, by contrast, τ_0,eff (temperature corrected) was found to increase by approx. 1.6- to 2-times in the temperature interval 35°C to 22°C, a result consistent with the dependence of the cold cataract phase transition temperature on γ-crystallin concentration. Analysis of intensity ratio dispersion curves, using an assumed model of isodesmic association, permitted the estimation of the association constant characterizing the aggregation under particular conditions of concentration and temperature. The significant increase in the value of the association constant with moderate increases in protein concentration was rationalized by invoking the effect of ‘macromolecular crowding’. The results obtained in this study suggest that in the intact lens, where high protein concentrations prevail, γ-crystallin is unlikely to be found in the monomeric state, but more likely, as a significantly aggregated species, representing a broad molecular weight distribution.     

On the electric polarization of DNA

Dielectric dispersion of DNA was studied in the frequency range 100 Hz–100 kHz at four different temperatures (6–30°C). The dielectric increment ε₀–ε_∞ increased with the rise of temperature. The relaxation time, on the other hand, decreased. Both the increase in dielectric increment and the decrease in relaxation time could not be explained on the basis of the counterion polarization theory. Dipole moment was estimated from Kirkwood theory. It was found to decrease systematically with temperature. Even at 0°C there was a dipole moment of 10⁴D.     

The mechanical properties of stratum corneum: I. The effect of water and ambient temperature on the tensile properties of newborn rat stratum corneum

The tensile properties of the outermost layer of skin of neonatal rats, the stratum corneum, were investigated at a constant strain rate as a function of moisture content and ambient test temperature. The results show that the mechanical behavior of this membrane, whose primary constituent is the fibrous protein keratin, can be significantly altered by variations in both the sorbed water content and ambient temperature. In particular, a brittle to ductile transition was observed at 25°C once the hydration level exceeded 70% relative humidity. Similarly, an identical phenomenon moisture concentrations were maintained at 10 g H₂O/100 g dry protein. Differential scanning calorimetry measurements showed the presence of a molecular relaxation process which migrated from 42°C at 40% relative humidity to −18°C at 95% relative humidity. It is postulated that this relaxation process, possibly corresponding to the glass transition of the fibrous protein component of stratum corneum, is primarily respnsible for the observed behavior.     

NMR study on molecular mobility of DNA molecules in solution

The molecular mobility of calf thymus DNA molecules in solution has been discussed in terms of correlation time τ calculated from measurements of longitudinal T₁ and transverse T₂ magnetic relaxation times. The influence of DNA concentration and ionic strength of the solution upon freedom of movement of DNA molecules was studied for native and denatured DNA and also during thermal helix-coil transition. The dependence of τ values on temperature was carried out by comparing the values of correlation times τ_tat given temperature with the correlation time τ₂₀ at 20°C. The molecular rotation of DNA at 20°C and at higher ionic strength at 0.15 and 1.0.M NaCl is described by τ values of the order of 1.0–1.2 × 10^?8 and was reduced slightly with increase of temperature below the helix-coil transition. The molecular rotation of DNA in 0.02MNaCl was lower at 20°C as compared to DNA in solvents with higher NaCl concentrations and increases rapidly with increase of temperature in the range 20–60°C. The values of correlation time are characterized by fast increase at temperatures above the spectrophotometrically determined beginning of melting curve. The beginning of this increase is observed at about 65, 80, and 85°C for DNA in 0.02, 0.15, and 1.0MNaCl, respectively. Values of correlation time for denatured DNA are in all cases about 1.1–1.4 times that for native DNA. The obtained results are discussed in terms of conformation of DNA molecules in solution as well as in terms of water dipole binding in DNA hydration shells.     

Effect of acute temperature transitions on chronotropic and inotropic responses of the South American lungfish Lepidosiren paradoxa

The in vivo and in vitro cardiac responses of Lepidosiren paradoxa were analyzed during temperature variations from 25°C (acclimation temperature) to 15°C and 35°C, and subsequent return to 25°C. Chronotropic (heart frequency) and inotropic (twitch force) responses varied directly with temperature, decreasing from 25°C to 15°C and increasing from 25°C to 35°C. However, time to peak tension (TPT) and time to half relaxation (THR) showed an inverse tendency. The results indicate that the myocardium of L. paradoxa responds more appropriately to acute elevations in temperature, which results in an increased cardiac performance due to both positive chronotropism and inotropism, in spite of the temperature-induced curtailment of TPT and THR.     

The low-temperature mechenical relaxation of elastin. I. The dry protein.

The low-temperature relaxation of native ox Ligamentum nuchae elastin and of two purified elastin samples has been investigated in the dry state by means of the Rheovibron DDV II viscoelastometer, at the fixed frequencies of 3.5, 11, 35, and 110 Hz. Besides the glass transition at about 200°C, a relatively strong secondary loss-tangent peak, whose activation energy is about 13 kcal/mol, is found for all samples below room temperature. The peak maximum is at ?71°C for the 3.5-Hz frequency. On the basis of its location, intensity, and width, it is attributed to a main-chain relaxation of the pure protein. When suitable reduced variables are used, the loss-tangent-temperature curves obtained at different frequencies can be satisfactorily superimposed to give a master curve. Comparison of the data with the prediction of a semiempirical viscoelastic model can also be considered satisfactory.     

Dielectric study of low-molecular weight mannan triacetate in chloroform

The permittivity ϵ′ and dielectric loss ϵ′' of low-molecular weight mannan triacetate in chloroform in the frequency range 1 kHz to 10 MHz at 20°C are reported. Deviations from a rod-like configuration are suggested to account for the observed behaviour of the dielectric relaxation time as a function of the degree of polymerization.     

Dielectric relaxation studies on bovine ligamentum nuchae

Dielectric relaxation studies of bovine ligamentum nuchae are reported over the frequency range of 1 MHz to 1 GHz and over the temperature range of 23–48°C. A temperature-dependent relaxation process was observed at low megahertz-frequency with the correlation time of around 40 ns. The result is quite similar to that of a synthetic polypentapeptide (VPGVG) and of α-elastin. The relaxation is proposed to arise in part from the peptide libration within the polypentapeptide of bovine ligamentum nuchae.     

Influence of temperature and ionic strength on the low-frequency dielectric dispersion of DNA solutions

The dielectric properties of DNA solutions at low frequencies (5 Hz to 2 kHz) have been measured by means of a four-terminal bridge method utilized to minimize electrode polarization errors. At 24°C native salt-free DNA has a very large specific dielectric increment, Δε/c = 9.8 × 10⁶ l/mol and a very low frequency relaxation centered at 18 Hz. Both the dielectric increment and the relaxation time are greatly decreased by partial heat denaturation at temperatures above 60°C or by addition of salt, the effects being much larger for divalent anions. These results are shown to be in qualitative agreement with theoretical treatments of counterion fluctuation polarization by McTague and Gibbs for the equilibrium case and by Mandel for relaxation. The ratio of the relaxation time for the low-frequency process to that previously observed at much higher frequencies suggests that these relaxations result from counterion fluctuations along the longitudinal and transverse axes of the molecule, respectively.     

Rigidity of myosin and myosin rod by electric birefringence

The rotational relaxation times of rabbit myosin and myosin rod have been determined by electric birefringence measurement. The relaxation time of myosin measured in 10 mM pyrophosphate buffers in a pH range of 7.6–9.5 was found to have substantial concentration and pH dependences. The infinite-dilution limit of the relaxation time, τ°, was determined as 38 ± 2 μs, and it was found to be independent of pH. For myosin rod, a possible thermally induced conformational change was investigated in a temperature range of 1–43°C. The rotational relaxation time of myosin rod shows no clear indication of conformational change in this temperature range, and the radius of gyration measurement by light scattering was shown to be consistent with this observation. The steady-state birefringence, however, decreases substantially above around 40°C. This, the myosin rod appears to be only slightly flexible even at physiological temperature, but the possibility of a “melting” or “hinging” of the myosin rod cannot completely be ruled out on the basis of these experiments.     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

On peculiarities of temperature dependences of water spectra in the terahertz frequency domain

We analyzed spectra of light and heavy water at temperatures from 4 up to 50°C in a frequency range of 0.15 to 6.5 THz. It was shown that the amplitude of high-frequency relaxation absorption band with its maximum at 0.5 THz extends with increasing temperature and this temperature dependence for light water has a marked feature at 35–40°C as a sharp growth. This fact is noteworthy because this range corresponds to physiological values of a body temperature of the warm-blooded organisms. At the same time, the analogous temperature dependence for heavy water in the considered temperature range lacks this particular feature. Thus, the water with its properties differs significantly not only from other fluids, but also from its own isotopologues.     

The effect of freeze rate,duration of phase transition,and warming rate on survival of frozen canine kidneys

The effect of cooling rate, warming rate, and duration of phase transition upon survival of frozen canine kidneys was investigated. In the present study, 11 kidneys out of 14 rapidly cooled (2–4 °C/min) to ?22 °C and thawed (70–110 °C/min) were viable following contralateral nephrectomy. The serum creatinine and BUN levels rose to a maximum of 8.4 and 30 mg%, respectively, on the eighth day post-contralateral nephrectomy. Average survival time was 10 days; however, two of the dogs in this group were allowed to survive, one for 3 months and one for over 2 years. Eight kidneys out of 16 slowly cooled (0.25–1.0 °C/min) and either rapidly or slowly warmed (20–30 °C/min) had function to produce small amounts of urine; however, they did not survive more than 5 days after contralateral nephrectomy.Cooling rates of 0.1 and 10 °C/min were too harmful to the kidney to have renal function after reimplantation.The minimum renal cell damage as assessed by LDH and GOT in the post-freeze perfusate was found in the 2–4 °C/min cooling rate following rapid warming (70 °–110 °C/min).Correlation of the duration of phase transition time to renal cell damage was linear for LDH and GOT (r = 0.93). This result suggests that the duration of phase transition time also is an important factor during the freezing process, affecting postthaw survival of canine kidneys.     

Flexibility of collagen determined from dilute solution viscoelastic measurements

The frequency dependences of the storage and loss shear moduli, G′ and G″, of pronase-treated collagen dissolved in acetate buffer at pH 4.0 were measured at 17.0°C by use of the Birnboim-Schrag multiple lumped resonator apparatus. Some of the solutions contained 70% glycerol. The infinite-dilution moduli were determined and compared with theoretical models for a rigid cylinder and a semiflexible rod. Only the latter could fit the data. A rotational time of 144 μs and a slowest flexural relaxation time of 21 μs, both reduced to water at 20°C, were determined from the fit. The intrinsic viscosity and rotational relaxation time correspond to a semiflexible rod with persistence length of about 170 nm and a Young's modulus of 4 × 10¹⁰ dyn/cm².