Phase behavior and glass transition of 1,2-dioleoylphosphatidylethanolamine (DOPE) dehydrated in the presence of sucrose

The effect of sucrose on the phase behavior of 1,2-dioleoylphosphatidylethanolamine (DOPE) as a function of hydration was studied using differential scanning calorimetry and X-ray diffraction. DOPE/sucrose/water dispersions were dehydrated at osmotic pressures (Pi) ranging from 2 to 300 MPa at 30 degrees C and 0 degrees C. The hexagonal II-to-lamellar gel (H(II)-->L(beta)) thermotropic phase transition was observed during cooling in mixtures dehydrated at Pior=57 MPa, the H(II)-->L(beta) thermotropic phase transition was precluded when sucrose entered the rigid glassy state while the lipid was in the H(II) phase. Sucrose also hindered the H(II)-to-lamellar crystalline (L(c)), and H(II)-to-inverted ribbon (P(delta)) lyotropic phase transitions, which occurred in pure DOPE. Although the L(c) phase was observed in dehydrated 2:1 (mole ratio) DOPE/sucrose mixtures, it did not form in mixtures with higher sucrose contents (1:1 and 1:2 mixtures). The impact of sucrose on formation of the ordered phases (i.e., the L(c), L(beta), and P(delta) phases) of DOPE was explained as a trapping of DOPE in a metastable H(II) phase due to increased viscosity of the sucrose matrix. In addition, a glass transition of DOPE in the H(II) phase was observed, which we believe is the first report of a glass transition in phospholipids.     

Limits to life at low temperatures and at reduced water contents and water activities

Liquid water is generally considered an absolute requisite for functional life; consequently, life is expected to function only over the range of temperatures that permit its existence. These limits, however, do not apply to cell survival. Some cells can survive the closest attainable approach to 0 K, and some can survive the loss of over 99% of their water.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.     

Thermal properties of water in myoglobin crystals and solutions at subzero temperatures.

The water of hydration in myoglobin crystals and solutions was studied at subzero temperatures by calorimetry and infrared spectroscopy (ir). For comparison we also investigated glycine, DL-alanine and DL-valine solutions. The hydration water remains amorphous at low temperatures. We find a broad glass transition between 180 and 270 K depending on the degree of hydration. The ice component shows a noncolligative melting point depression that is attributed to a finite conformational flexibility. The ir spectrum and the specific heat of water in myoglobin crystals was determined for the first time between 180 and 290 K. The glass transition in crystals is qualitatively similar to what is found in amorphous samples at the same water content. These data are compared with M?ssbauer experiments and dielectric relaxation of water in myoglobin crystals. The similar temperature dependencies suggest a cross correlation between structural fluctuations and the thermal motion of crystal water. A hydrogen bond network model is proposed to explain these features. The essential ingredients are cooperativity and a distribution of hydrogen-bonded clusters.     

Kinetics of water loss from cells at subzero centigrade temperatures

A mathematical model is developed for the calculation of the kinetics of water loss from cells at subzero centigrade temperatures. In this model it is assumed that the cell surface membrane is permeable to water only, the protoplasm is a nonideal solution, the cells are spherical, and during the cooling process the cell temperature is not uniform inside the cell. It is also assumed that because of water loss due to cooling process the cell volume and the cell surface area reduce and the reductions in surface area and volume of the cell are functions of the amount of water loss from the cell. Based on this model, and for different conditions, the fractions of supercooled intracellular water remaining in the cells at various temperatures are calculated.It is shown that for cooling cells at subzero centigrade temperatures. (1) the consideration of Clausius-Clapeyron equation for vapor pressures of water and ice, instead of the exact vapor pressure relations, may produce errors in the prediction of the amount of water loss from the cells at high cooling rates only, (2) the assumption of intact cells will produce considerable deviation in the prediction of water loss from the cells as compared to the more realistic assumption of shrinkable cells, (3) the nonideality of protoplasm solution is very effective on the prediction of the amount of water loss from the cells, and (4) the assumption of uniform-temperature cells during the cooling process may be erroneous only for cells with small fractions of water in their protoplasms.     

Fast photochemical reactions of cytochrome P450 at subzero temperatures.

Several reactions of the cytochrome P450 multi-step cycle have been studied by fast light activation combined with subzero temperatures. A flash device was adapted to an Aminco-Chance DW 2 spectrophotometer equipped for subzero temperature thermostatisation. The first electron can be introduced into the cycle by non specific reducing agents such as reduced flavin mononucleotide (FMNH2) or methylviologen radical (MV.). This first reduction remains a fast process even at subzero temperatures. The oxy-compound Fe2+-O2 can thus be formed either directly from Fe2+ or via the photodissociation of the carboxy-ferro adduct. Fe2+-O2 is stable at subzero temperatures towards spontaneous autoxidation as well as further reduction by FMNH2 or MW.. In addition, the recombination of CO after flash photodissociation of Fe2+-CO was used to study in more details the specific behaviors of the purified microsomal cytochrome.     

Hybrid formation for liganded hemoglobins A and C at subzero temperatures.

The kinetics of formation of the asymmetric carbonmonoxyhemoglobin hybrid (alpha beta)A(alpha beta)C from the parent molecules alpha 2 beta 2A and alpha 2 beta 2C have been studied by electrophoresis at subzero temperatures (down to -40 degrees C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH greater than 10, t = -40 degrees C, the hybrid between alpha 2 beta 2A and alpha 2 beta 2C is stable for several hours. A rapid thermal quenching of a mixture of alpha 2 beta 2A and alpha 2 beta 2C prevented hybrid formation during the time required to separate the 2 molecules.     

Membrane energization at subzero temperatures: calcium uptake and oxonol-V responses

The use of aprotic solvents for preserving the electron transport properties of mitochondria at subzero temperatures is based upon the use of binary water and ethylene glycol mixtures or upon ternary and quaternary mixtures that include dimethyl sulfoxide and the lower aliphatic alcohols. In order to better preserve the respiratory control properties of mitochondria at subzero temperatures, detailed studies have been made of the effects of these mixtures on the respiratory control and electron transport from NADH or succinate of mitochondrial preparations. It is found that ADP is not metabolized at a measurable rate below 0 °C, but that Ca²⁺ is rapidly taken up and can thus be used to assay respiratory control ratios down to ?8 °C. In the region below ?8 °C the charge-sensitive probe oxonol-V has been used to evaluate energy coupling. By using Ca²⁺ to stimulate respiration at 0 °C good results are obtained with ethylene glycol/water alone and optimal results are obtained with a quaternary mixture. A mixture that freezes at ?21 °C gives about 50% inhibition of the respiratory control ratio for electron transport at 0 °C with NADH or succinate as substrates. The mixtures permit low-temperature studies of mitochondrial functions under conditions of minimal respiratory rate, including the kinetics of electron transfer reactions, the formation of intermediate compounds, and the rapid freeze-trapping of mitochondrial reactions for analytical chemistry or ³¹P NMR.     

Behavioral responses of hatchling painted turtles (Chrysemys picta) and snapping turtles (Chelydra serpentina) at subzero temperatures

We monitored behavioral responses of cold-acclimated hatchling painted turtles (Chrysemys picta) indigenous to Nebraska and hatchling snapping turtles (Chelydra serpentina) indigenous to Nebraska and Arkansas during cooling (0.1°C/min) to temperatures as low as −19°C. All turtles made exploratory movements during cooling and locomotion occurred at temperatures as low as −2 to −4°C, but C. picta maintained relatively higher levels of locomotor activity than C. serpentina, and no differences in motility occurred between northern and southern groups of C. serpentina. Slow movements of the head and limbs were observed in supercooled hatchling C. picta at temperatures as low as −10°C, whereas at about −5°C, C. serpentina exhibited an increase in spontaneous motor activity followed by muscle contracture, immobility, and spontaneous freezing. C. picta spontaneously froze at about −16°C without exhibiting cold contracture, suggesting that they are better adapted to survive exposure to extreme cold.     

The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate.

The reaction of β-galactosidase ($\text{E. coli}$ K12) with $\text{o}$-nitrophenyl-β-$\text{D}$-galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of $\text{o}$-nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with E_a = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on K_m but caused a small decrease in K_cat.     

Structure and dynamics of primary hydration shell of phosphatidylcholine bilayers at subzero temperatures.

Deuterium NMR relaxation and intensity measurements of the 2H-labeled H2O/dimyristoyl phosphatidylcholine bilayer were performed to understand the molecular origin of the freezing event of phospholipid headgroup and the structure and dynamics of unfrozen water molecules in the interbilayer space at subzero temperatures. The results suggest that about one to two water molecules associated with the phosphate group freeze during the freezing event of phospholipid headgroups, whereas about five to six waters near the trimethylammonium group behave as a water cluster and remain unfrozen at temperatures as low as -70 degrees C. In addition, temperature-dependent T1 and T2 relaxation times suggest that dynamic coupling occurs not only between the phosphate group and its bound water, but also between the methyl group and the adjacent water molecules. Based on these observations, the primary hydration shell of phosphatidylcholine headgroup at subzero temperatures is suggested to consist of two distinct regions: a clathrate-like water cluster, most likely a water pentamer, near the hydrophobic methyl group, and hydration water molecules associated with the phosphate group.     

High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures

High hydrostatic pressure is a new technology in the food processing industry, and is used for cold pasteurization of food products. However, the pressure inactivation of food-borne microorganisms requires very high pressures (generally more than 400 MPa) and long pressure holding times (5 min or more). Carrying out pressure processing at low temperatures without freezing can reduce these parameters, which presently limit the application of this technology, in keeping the quality of fresh raw product. The yeast, Saccharomyces cerevisiae and the bacterium, Lactobacillus plantarum were pressurized for 10 min at temperatures between -20 and 25 degrees C and pressure between 100 and 350 MPa. Pressurization at subzero temperatures without freezing significantly enhanced the effect of pressure. For example, at a pressure of 150 MPa, the decrease in temperature from ambient to -20 degrees C allowed an increase in the pressure-induced inactivation from less than 1 log up to 7-8 log for each microorganism studied. However, for comparable inactivation levels, the kinetics of microorganism inactivation did not differ, which suggests identical inactivation mechanisms. Implications of water thermodynamical properties like compression, protein denaturation, as well as membrane phase transitions, are discussed.     

Survival of emu (Dromaius novaehollandiae) sperm preserved at subzero temperatures and different cryoprotectant concentrations

Three experiments were conducted to optimize the protocol for cryopreservation of emu sperm. Ejaculates were collected from trained male emus then diluted 1:1 and pooled before allocation to treatments and measured for sperm viability, motility, egg membrane penetration ability, membrane stability, and morphology. In Experiment 1, semen was either cooled to 5 °C after dilution or diluted with a precooled to 5 °C diluent before cooling to 5 °C and then frozen at liquid nitrogen vapor temperatures of −140 °C and −35 °C, with 6% or 9% dimethylacetmide (DMA; a permeating cryoprotectant) and compared for sperm functions. The percentages of viable (42.8 ± 1.1%), normal (39.0 ± 1.3%), and motile (29.8 ± 1.3%) sperm were higher (P < 0.001) for semen frozen at −14 °C with 9% DMA (path 2) than for all other combinations. In Experiment 2, we assessed the value of combining DMA and trehalose in the diluent. Combining trehalose (3% to 9%) with DMA (3% to 9%) prior to freezing reduced (P < 0.001) the percentages of postthaw viable (by 4 to 9 ± 1.2%), normal (by 5 to 11 ± 1.3%), and motile sperm (by 13 to 17 ± 2.5%) and the number of holes on the perivitelline layer (by 27 to 29 holes/mm²). Postthaw function was best preserved with 9% DMA alone. In experiment 3, we investigated the possibility of increasing DMA concentrations from 6% to 24%. Postthaw sperm viability (52 to 55 ± 2.3%) and morphology (48 to 51 ± 1.7%) were higher (P < 0.05) with 18% and 24% than with 6% to 12% DMA and did not differ between 18% and 24% DMA. However, sperm motility (36 to 43 ± 2.9%) and the number of perivitelline holes were similar (P > 0.05) for 9% to 18% DMA (36 to 55 ± 12%). We concluded that adding 6% to 9% trehalose to the diluent offered no advantage, and that the current best practice for preserving postthaw function in emu sperm is to dilute semen with a precooled to 5 °C diluent and use 18% DMA.     

Hydraulic permeability and activation energy of human keratinocytes at subzero temperatures

Studies on isolated human keratinocytes provide a model for design of optimal freeze-thaw protocols for skin cryopreservation and banking. Nucleated keratinocytes from the basal layer of split thickness human cadaveric skin were separated by a combined trypsin and DNAse digestion and suspended in Dulbecco's minimal essential medium with fetal calf serum. A small volume of suspension was frozen on a microprocessor controlled cryostage. Extracellular ice was nucleated at predetermined subzero temperatures, and the temperature was held constant for the duration of the experiment. The osmotic response of the cells to the formation of extracellular ice was recorded on 35-mm photographic film. Selected serial frames were digitized for automated computer evaluation of metric parameters of specific cells. Changes in the apparent cell volume were quantified over a period of several minutes to obtain dehydration curves associated with exposure to concentrated extracellular electrolytes. The Kedem-Katchalsky coupled flow transport model was statistically fit to the data using a personal computer. Values for the permeability coefficients were adjusted to optimize the correlation between the theory and the data. An activation energy of 44.8 kJ/mol and a water permeability of 0.035 micron (atm.min) at 0 degrees C were derived from the data measured over a temperature range from -2 to -9 degrees C.     

Analysis of the water permeability of human granulocytes at subzero temperatures in the presence of extracellular ice

The plasma membrane water permeability of human granulocytes in the presence of extracellular ice was determined experimentally on a cryomicroscope. Transient volumes of individual cells were measured at constant subzero temperatures subsequent to ice nucleation. Permeability values were deduced by adjustment of multiple parameters in a model to obtain an optimal fit to the data. The permeability was determined to be a function of both temperature and intracellular solute osmolality, with a reference value at 0 degrees C of 0.407 micrometers/atm.min and temperature and solute coefficients of 218kJ/mol and 1.09 Osm/kg.