Compartmentalization of NaCl in frozen solutions of antifreeze glycoproteins.

The freezing behavior of NaCl solutions containing antifreeze glycoproteins from an Antarctic fish has been investigated to determine whether the glycoproteins prevent concentration of NaCl during freezing. Frozen NaCl solutions containing glycoproteins exhibit greater resistance to releasing their brine during centrifugation than NaCl solutions containing other cryoprotectants. With the aid of calorimetry this is shown to be caused not by an incorporation of the NaCl into the ice but by compartmentalization of the brine pockets. The compartmentalization is attributed to an unusual spicular structure that is imposed on the ice by glycoproteins.     

Aggregation of antifreeze glycoprotein fraction 8 and its effect on antifreeze activity

Antifreeze glycoproteins (AFGPs) have many potential applications ranging from the cryopreservation and hypothermic storage of tissues and organs to the preservation of various frozen food products. Since supplying native AFGP for these applications is a labor-intensive and costly process, the rational design and synthesis of functional AFGP analogues is a very attractive alternative. While structure-function studies have implicated specific structural motifs as essential for antifreeze activity in AFGP, the relationship between solution conformation and antifreeze activity is poorly understood. Toward this end, we have analyzed AFGP8 in aqueous solutions using dynamic light scattering (DLS) and circular dichroism (CD). Our results indicate that AFGP8 forms discrete aggregates in solution. These aggregates are predominantly composed of dimers that form at solution concentrations greater than 20 mM. CD spectroscopy indicates that the preferred solution conformation of AFGP8 is consistent with that of random coil. However, significant beta-sheet and alpha-helix character is observed in more concentrated solutions, indicating that these glycopeptides are highly flexible in solution. Aggregation appears to have a minimal effect on the overall solution conformation. Thermal hysteresis (TH) activity of the aggregated solutions is much higher than that of less concentrated solutions that do not form aggregates. While cooperative functioning between lower and higher molecular weight AFGPs has been reported, this is the first instance where cooperative functioning in lower molecular weight AFGPs has been observed.     

Study of alcohol binding with serum albumin by NMR spectra of frozen aqueous solutions

Interaction of alcohols (methanol, glycerol, sorbitol) with human serum albumin (HSA) was studied by the use of NMR spectroscopy in frozen aqueous solutions. The amount of binding was shown to increase in the series methanol-glycerol-sorbitol, on this basis a suggestion is made that interaction of alcohols with HSA occurs due to the formation of hydrogen bonds in which the hydroxyl groups act as proton donors. It is found that the presence of albumin influences significantly the outcome of eutectics from a diluted methanol solution and glycerol solution, the temperature of eutectics decreases in case of methanol and increases in case of glycerol.     

Volatile transport in frozen aqueous solutions. I. Development of a mechanism

Frozen aqueous butanol solutions are equilibrated at constant subzero temperature over activated charcoal. A fraction of the butanol is lost within 24 hr, the remainder being retained for over 350 hr. The retained butanol is lost only with the simultaneous loss of water. Pure ice is demonstrated to be permeable to the transport of butanol. Based on experiments which remove, the free surface, the butanol loss that is independent of water loss is shown to originate from a surface layer postulated to form during freezing of the solution.Three types of butanol-ice interactions are postulated: (1) Butanol in the surface layer; (2) butanol entrapped in interdendritic spaces; (3) butanol present in pores and cracks after sorption from the vapor state.     

Low-frequency Raman spectra in water and in weak aqueous solutions. Spatial inhomogeneity in hydrogen peroxide solution

We describe some examples of the study of intermolecular interactions in water and in weak aqueous solutions using the low-frequency Raman spectra. An improved method of determining parameters of the dynamic susceptibility from the Raman spectra is described. The nine spectral parameters (the intensity, the frequency and the width of the two oscillators, the intensity and the width of the relaxation motion and the intensity of fluorescent background) completely describe the spectrum in the frequency range of 4 to 320 cm^?1. For hydrogen peroxide the dependences of these parameters on the concentration is shown. Concentration dependence of the spectral parameters of the hydrogen peroxide solution in water indicates a change in the structure of the molecular bonds in water near a peroxide molecule at a distance up to 0.7–0.9 nm. The effect of the exciting laser radiation on the parameters of the spectra in the registration of the Raman spectra of water is shown.     

Volatile transport in frozen aqueous solutions. II. Influence of system parameters

Frozen aqueous butanol solutions are evaluated for the influence of sample preparation and equilibration conditions on the loss of butanol.It is shown that an increase of equilibration temperature results in increased butanol loss, both water independent and water dependent. The freezing rate will influence the equilibration retention level, with faster freezing resulting in smaller butanol loss. An increase in butanol concentration gave the same percentage of butanol loss, i.e., the amount of butanol lost was directly proportional to the initial concentration.     

Measurement of grain growth in the recrystallization of rapidly frozen solutions of antifreeze glycoproteins

A quantitative estimate of the activation energy for grain growth has been obtained by analyzing ice recrystallization experiments from water and from solutions with small amounts (< 1.0 μg/mL) of antifreeze glycoprotein (AFGP). Rates of grain growth are measured as changes of grain diameter in time, with the supercooled holding temperature aVid glycoprotein concentration as parameters. Arrhenius plots of these rates vs (1/T) yielded slopes proportional to the activation energies for the particular species. The values of activation energy are almost independent of solution concentration or the species of AFGP. Averaged activation energy value for the AFGP-4 species is Q_g = (6.61 ± 1.02) × 10⁵ J/mole. The “less active” AFGP-8 yielded an average Q_g = (5.71 ± 2.39) × 10⁵ J/mole, quite similar to the AFGP-4 species. The activation energy for recrystallization in a pure ice-water system was estimated from two temperature points, T = ?5.4 and ?7.5°C. The best value is 2.39 × 10⁵ J/mole, nearly twice that obtained by M. N. Martino and N. E. Zaritsky [(1989) Cryobiology, Vol. 26, p. 138] in a recrystallization experiment using salt solution, but much smaller than the values derived from the AFGP solutions. Results further show that activation entropy is at least a factor of 2 larger for the AFGP species than that of pure ice-water system under the same growth conditions. These results suggest significant roles, both energetically and entropically, for AFGP molecules in their ability to inhibit grain growth of ice. © 1994 John Wiley & Sons, Inc.     

Effects of a type I antifreeze protein (AFP) on the melting of frozen AFP and AFP+solute aqueous solutions studied by NMR microimaging experiment

The effects of a type I AFP on the bulk melting of frozen AFP solutions and frozen AFP+solute solutions were studied through an NMR microimaging experiment. The solutes studied include sodium chloride and glucose and the amino acids alanine, threonine, arginine, and aspartic acid. We found that the AFP is able to induce the bulk melting of the frozen AFP solutions at temperatures lower than 0 °C and can also keep the ice melted at higher temperatures in the AFP+solute solutions than those in the corresponding solute solutions. The latter shows that the ice phases were in super-heated states in the frozen AFP+solute solutions. We have tried to understand the first experimental phenomenon via the recent theoretical prediction that type I AFP can induce the local melting of ice upon adsorption to ice surfaces. The latter experimental phenomenon was explained with the hypothesis that the adsorption of AFP to ice surfaces introduces a less hydrophilic water-AFP-ice interfacial region, which repels the ionic/hydrophilic solutes. Thus, this interfacial region formed an intermediate chemical potential layer between the water phase and the ice phase, which prevented the transfer of water from the ice phase to the water phase. We have also attempted to understand the significance of the observed melting phenomena to the survival of organisms that express AFPs over cold winters.     

Electron paramagnetic resonance in biochemistry. Computer simulation of spectra from frozen aqueous samples.

Two computer programs are described; they can be used to simulate e.p.r. spectra of effective spin-1/2 systems in frozen aqueous samples. One program is written in BASIC and the other in FORTRAN IV. Both programs are deposited as a Supplementary Publication (SUP 50082; 27 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K. References are given to the applications of these programs in biochemical work.     

Laser Raman spectra of calf thymus chromatin and its constituents.

Extensive Raman measurements have been made on calf thymus chromatin, core chromatin, the (H3,H4)/DNA complex, and isolated DNA. The results indicate that the alpha-helical content of the nucleosomal histones gradually increases as they form the heterocomplexes that lead to the formation of the octameric nucleosome core. The secondary structure of the latter is not modified as it binds to DNA. The spectra indicate that the DNA essentially retains its B conformation in nucleosomes, although slight changes probably occur in the ribose-phosphate backbone. No specific interactions between the nucleosomal histones and DNA can be established from the spectra, but histone H1 possibly interacts selectively with the thymine bases.     

Final products obtained from the gamma radiolysis of frozen aqueous solutions of thymidine.

The final radiation products obtained by gamma-irradiation of frozen aqueous solutions of thymidine have been identified as 5,6-dihydro-5,6-dihydroxythy-midine, 5,6-dihydrothymidine, thymidine dimers, 1-(2-deoxy-beta-D-threo-pento-furanosyl)-thymine, 1-(2-deoxy-alpha-L-threo-pentofuranosyl)-thymine, thymine and 5,6-dihydrothymine. The nature of the radiation products could be explained on the basis of the radical structures reported earlier.     

Computer analysis of anisotropic ESR spectra and the structure of radical H and OH adducts of nucleic acid components in frozen aqueous solutions

Computer analysis of ESR spectra recorded under gamma-irradiation of polycrystal and glass-like solutions of nucleotides and nitrous bases was carried out to determine the localization sites of free valency (damage) in DNA macromolecule when interacting with water radiolysis products (OH and H). The analysis was performed on a mini-computer with the program of reconstruction of ESR anisotropic spectra in the variation search regime. When calculating the theoretical spectra anisotropic width of the lines, g-factors and HHS constants on X and Z axes were taken into account. Good reproduction of the experimental spectra on the computer was achieved, as a result the ESR spectra were obtained. Relative contribution of each of OH- and H-adducts of nitrous bases to the total ESR spectrum of gamma-irradiated DNA will be determined in future.