Felix Franks

Polymer cryoprotectants in the preservation of biological ultrastructure. I. Low temperature states of aqueous solutions of hydrophilic polymers.

The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoproteetants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspherical morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoprotectants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspheral morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.

The Properties of Aqueous Solutions at Subzero Temperatures

The past 15 years have witnessed an increasing interest in the many changes which accompany the freezing and thawing of aqueous systems. Even so, most of the experimental and theoretical advances during that period have been achieved against a background of general unawareness by those who are the would-be exploiters of such knowledge. This chapter is an account of the physical and chemical changes undergone by aqueous systems at low temperatures, in the frozen or nonfrozen state. Some biological implications are also considered.

Polymeric cryoproteetants in the preservation of biological ultrastructure

A study has been made of the physiological effects of three non‐penetrating polymeric cryoprotective agents on sixteen different plant and animal cells and tissues. The cryoproteetants, when used at concentrations at which they are effective in preventing ice‐crystal formation, generally have a lower toxicity to cells and tissue than similar concentrations of glycerol. The relatively low toxicity of these substances suggests that they would be more suitable as cryoproteetants for morphological and analytical studies than the commonly used low molecular weight compounds.

Salt precipitation during the freeze-concentration of phosphate buffer solutions

Salt precipitation during the freeze concentration of phosphate solutions was investigated by differential scanning calorimetry (DSC), in view of its practical importance in the cryopreservation or freeze-drying of biological materials. It was found that the fraction of salt precipitated depends on the initial salt concentration; it began to decrease with decreasing concentration at approx. 1 M. Salt precipitation also depends on the cooling rate. In some cases, cooling at approx. 10(3) degree min-1 inhibited salt precipitation which had been observed during slow cooling (0.62 degree min-1), without, however, affecting the shape of the ice melting endotherm. In the case of ternary phosphate buffers, the fraction of salt precipitating depends on the salt composition as well as the initial concentration and cooling rate. Near the composition of the ternary eutectic or the composition where two salts are present at the same concentration, salts were prevented from precipitation.

Ice nucleation and freezing in undercooled cells

DSC has been employed to study the effect of cooling on a range of cells under exclusion of extracellular ice and in the absence of chemical cryoprotectants. In contrast to earlier reports, all the cells studied were found to freeze at temperatures above that indicated for homogeneous nucleation of ice in undercooled liquid water. In the case of human erythrocytes this temperature difference was only 0.5 degrees, but for yeast cells and cells of plant origin the difference amounted to congruent to 9 degrees. Nucleation of ice within the cell (or at the cell wall/membrane) must therefore be initiated by a heterogeneous mechanism. A kinetic analysis of the temperature dependence of nucleation shows the rates to be consistent with the dimensions of the plant cells (or organelles), if these were to be the active nucleators. However, the nucleation kinetics of human erythrocytes are extremely temperature sensitive, and the kinetic parameters only differ by small, though significant, extents from those of the suspension medium. Possible nucleation mechanisms are discussed in terms of the experimental data and the cell dimensions. Finally, one of the underlying assumptions of the kinetic analysis, i.e., that ice growth must be rapid compared to nucleation, has been tested and validated by freeze-fracture electron microscopy.

Nucleation rates of ice in undercooled water and aqueous solutions of polyethylene glycol

Abstract Differential calorimetry has been employed in two different instrumental modes to measure ice nucleation rates in undercooled water and aqueous solutions of polyethylene glycol (PEG) as a function of temperature. The results are consistent with the classical theory of homogeneous nucleation kinetics. It appears that the inhibition of ice nucleation by PEG results mainly from a marked perturbation of the diffusional freedom of water molecules by the polymer.

Solute—Water interactions: Do polyhydroxy compounds alter the properties of water?☆

The interactions between PHCs and water, like those between water molecules, are governed by hydrogen bonding. The details of these interactions are very sensitive to spacings and orientations of the -OH groups on the solute molecules. Where different conformers can coexist in solution, the aqueous solvent acts so as to favor the conformer with the largest number of equatorial -OH groups, because of their spatial compatibility with water. Because of this compatibility, aqueous solutions of PHCs have the tendency to supersaturation and incomplete freezing, manifestations of the phenomenon known as bound water which is, however, a misnomer. The range of water structure perturbation is probably governed by hydration forces which appear to dominate at solute-solute distances of less than 3 nm and which decay exponentially. Although on a single hydrogen bond basis the hydration effects are marginal, they nevertheless are responsible for many macroscopic phenomena, e.g., gel formation, liquid crystals, and protection against dehydration.

Cold-lability of phosphofructokinase from potato tubers

Abstract The aim of this work was to study the cold-lability of phosphofructokinase from tubers of Solanum tuberosum cv Record, a variety that exhibits low temperature sweetening. The enzyme was purified by affinity chromatography and samples were examined by differential scanning calorimetry. Power-time curves were recorded for cooling and warming between 293 and 265 K. This revealed an exothermic dissociation, centred on 286 K, as the temperature was lowered. The latter temperature is close to that at which the tubers start to sweeten. It is suggested that hydrophobic interactions that contribute to the stability of the active configuration of the oligomeric enzyme are weakened at low temperatures, and that this causes spontaneous dissociation and consequent loss of activity of the enzyme. The results are discussed in relation to low temperature sweetening of potatoes.

The cold-induced denaturation of lactate dehydrogenase at sub-zero temperatures in the absence of perturbants

Lactate dehydrogenase; Enzyme denaturation, cold; pH; Temperature; Cryosolvent

Blood glycoprotein from antarctic fish possible conformational origin of antifreeze activity

High resolution 1H NMR and circular dichroism (CD) measurements have been performed on aqueous solutions of antarctic fish antifreeze glycoprotein. The carbohydrate contribution ot the observed CD spectrum has been estimated from closely analogous model compounds. The residual peptide contribution cannot be interpreted of the known spectral behaviour of alpha-helix, beta-sheet and random coil. Instead it resembles the CD spectrum of beta-structure in position, magnitude and spectral form, but is of opposite sign, indicating a specific but unusual peptide conformation, which we suggest may be stabilised by non-bonded interactions between the peptide backbone and the carbohydrate sidechains. Previous evidence which supports this interpretation is reviewed. NMR and CD measurements between -2 and +30 degrees C are consistent with conformational stability throughout the biologically relevant temperature range. The mechanism of the antifreeze activity is discussed in terms of the spatial and orientational correlations of sugar hydroxy groups and water in the liquid and solid states. The implication of an ordered peptide structure is explained by the comparison of the antifreeze glycoprotein with synthetic water-soluble polymers which also exhibit limited antifreeze properties.