Thermodynamic aspects of vitrification

Vitrification is a process in which a liquid begins to behave as a solid during cooling without any substantial change in molecular arrangement or thermodynamic state variables. The physical phenomenon of vitrification is relevant to both cryopreservation by freezing, in which cells survive in glass between ice crystals, and cryopreservation by vitrification in which a whole sample is vitrified. The change from liquid to solid behavior is called the glass transition. It is coincident with liquid viscosity reaching 10¹³ Poise during cooling, which corresponds to a shear stress relaxation time of several minutes. The glass transition can be understood on a molecular level as a loss of rotational and translational degrees of freedom over a particular measurement timescale, leaving only bond vibration within a fixed molecular structure. Reduced freedom of molecular movement results in decreased heat capacity and thermal expansivity in glass relative to the liquid state. In cryoprotectant solutions, the change from liquid to solid properties happens over a ∼10 °C temperature interval centered on a glass transition temperature, typically near −120 °C (±10 °C) for solutions used for vitrification. Loss of freedom to quickly rearrange molecular position causes liquids to depart from thermodynamic equilibrium as they turn into a glass during vitrification. Residual molecular mobility below the glass transition temperature allows glass to very slowly contract, release heat, and decrease entropy during relaxation toward equilibrium. Although diffusion is practically non-existent below the glass transition temperature, small local movements of molecules related to relaxation have consequences for cryobiology. In particular, ice nucleation in supercooled vitrification solutions occurs at remarkable speed until at least 15 °C below the glass transition temperature.     

Introduction to the special issue: Thermodynamic aspects of cryobiology

This brief paper introduces the subject of thermodynamics and the papers of the special issue on thermodynamic aspects of cryobiology. Thermodynamic terminology is defined for the non-specialist.     

Refinement of the structure of beta-chitin.

The structure of β-chitin has been refined by rigid-body least-squares methods, based on the intensity data for highly crystalline specimens from the pogonophore Oligobrachia ivanovi. The structure consists of an array of poly-N-acetyl-D -glucosamine chains all having the same sense, which are linked together in sheets by N? H … O?C hydrogen bonding of the amide groups. In addition to the O-3′? H … O-5 intramolecular hydrogen bond, analogous to that in cellulose, the CH₂OH side chain forms an intrasheet hydrogen bond to the carbonyl oxygen on the next chain. This structure shows considerably better agreement between observed and calculated intensities than that possessing an intersheet hydrogen bond, as had been proposed previously. The structure is consistent with the swelling properties of β-chitin and can also be seen to be analogous to that of native cellulose.     

The deacetylation reaction in Eucalyptus wood: Kinetics and effects on the effective diffusion

The removal of native acetyl groups from hardwood O-acetyl-glucuronoxylan has a strong effect on physical characteristics, accessibility and structure of this polymer. The removal also has effects on the swelling and ion transport capacity of the cell wall of hardwoods. In this work, a kinetic expression for Eucalyptus wood deacetylation is determined. Two liquid mediums are considered: a simple alkaline one and another with a higher sodium concentration. The kinetic expression is a power law for the acetyl content and the concentrations in the liquid medium dependence, and is an Arrhenius type expression for temperature dependence. The kinetic expression can be useful to predict the physical properties of wood since the analysis of deacetylation effects on effective capillarity (ECCSA) shows that the acetyl content is a determining factor of wood ionic transport capacity.     

Comparative study of the first heterogeneous deacetylation of alpha- and beta-chitins in a multistep process

Heterogeneous deacetylation of alpha- and beta-chitins from shrimp shells and squid pens were comparatively studied. Each deacetylated sample, recovered after neutralization, was fractionated according to a water-soluble and insoluble fraction (pH 8.5). The systematic study of DAs, crystallinity changes, and distribution of N-acetylglucosamine residues were performed on the two kinds of fractions. For the two fractions resulting from a deacetylation in the presence of 50% (w/v) NaOH, for temperatures ranging from 80 to 110 degrees C, the activation energies of the reactions were found close to 39.9 +/- 1.0 and 42.8 +/- 1.8 kJ mol(-1) and the frequency factors of collision were of 7.2 +/- 2.4 and 54.4 +/- 18.5 10(3) min(-1), for alpha- and beta-chitins, respectively. Deacetylations of water-soluble and insoluble fractions were compared, and the major role played by the crystallinity level during deacetylation was evidenced and, thereafter, the role of the nature of the starting chitins on the chemical behavior. Thus, in some conditions, we observed critical values of DDA where the structures were becoming fully amorphous.     

Structural insights into intermediate steps in the Sir2 deacetylation reaction

Sirtuin enzymes comprise a unique class of NAD(+)-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD(+) analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.     

Thermodynamic aspects of DsbD-mediated electron transport

DsbD from Escherichia coli transports electrons from cytoplasmic thioredoxin across the inner membrane to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of three domains: a periplasmic N-terminal domain, a central transmembrane domain (tmDsbD) and a periplasmic C-terminal domain. Each domain contains two essential cysteine residues that are required for electron transport. In contrast to the quinone reductase DsbB, HPLC analysis of the methanol/hexane extracts of purified DsbD revealed no presence of quinones, suggesting that the tmDsbD interacts with thioredoxin and the periplasmic C-terminal domain exclusively via disulfide exchange. We also demonstrate that a DsbD variant containing only the redox-active cysteine pair C163 and C285 in tmDsbD, reconstituted into liposomes, has a redox potential of − 0.246 V. The results show that all steps in the DsbD-mediated electron flow are thermodynamically favorable.     

Formation of a lamellar compound by reaction of acrylic acid crystallosolvated in highly crystalline beta-chitin

A lamellar compound resulted from reaction of acrylic acid inside crystalline beta-chitin and the structure was investigated. Beta-chitin acts like a layered crystal, having stacked molecular sheets composed of parallel chains bound in one direction by intermolecular amide hydrogen bonding. Small guest molecules can be inserted between the molecular sheets, and a crystallosolvate can be formed. By immersion of beta-chitin in acrylic acid, a crystallosolvate was formed, which was then changed into the more stable lamellar compound by heat treatment at 105 degrees C. NMR measurement and IR spectroscopy showed that during the heat treatment there was a reaction between acrylic acid and the beta-chitin molecular sheet, but the sheet structure was maintained. By IR with deuteration, it was shown that the accessibility of solvents to this lamellar compound was greater than that for the initial beta-chitin. The lamellar compound is considered a kind of "pillared" structure related to the lamellar crystal.     

Structural data on the intra-crystalline swelling of beta-chitin

The intra-crystalline swelling of the highly crystalline beta-chitin from Tevnia jerichonana was investigated by X-ray crystallography and Fourier transform infrared (FTIR) spectroscopy, using hydrogenated and deuterated hydrochloric acids as swelling agents. Three levels of swelling were identified that could be defined as inter- and intra-sheet swelling. A moderate and reversible swelling in water and methanol gave crystalline beta-chitin cystallosolvates, namely dihydrate and methanolate, respectively. In these, an inter-sheet swelling was observed, corresponding to an expansion of only the b parameter of the unit cell of beta-chitin. Under these swelling conditions, the use of deuterated reagents had no effect on the amide N&z.sbnd;H⋯O&z.dbnd6;C hydrogen bonds that hold the structure of beta-chitin together, but only induced a partial and reversible deuteration of the chitin hydroxymethyl groups. A more severe swelling - but still reversible - occurred with 6 N HCl or DCl, which converted the crystals of beta-chitin into a paracrystalline gel-like product resulting from inter-sheet+intra-sheet swelling. With this acid strength, the deuteration pattern indicated that a fraction of the amide hydrogen bonds was broken and became susceptible to an irreversible deuteration. A very severe and irreversible swelling occurred with 8 N HCl or DCl. In that case, the inter- and intra-sheet swelling was extensive to the point where all memory of the parallel-chain beta-chitin was lost. In addition, this swelling was accompanied by a drastic and rapid depolymerization. The treatment with 8 N HCl led invariably to crystalline alpha-chitin when the samples were neutralized.     

Thermodynamic aspects of the CO-binding reaction to cytochrome P-450cam. Relevance with their biological significance and structure

The CO-binding kinetics of cytochrome P-450_cam(+) and P-450cam(−) have been measured in the millisecond time domain using a flash photolysis method. We have determined the reaction coordinates for free energy, enthalpy and entropy from the temperature dependence of the overall rate constants of the bimolecular forward (on) and backward (off) reactions. Comparing the thermodynamic profiles of P-450_cam with that of myoglobin (Mb) reported so far, the enthalpy and the entropy coordinates exhibit the following remarkable characteristics. The CO-binding equilibrium: The stability of the CO-complex is perfectly entropy-driven for P-450_cam, while enthalpy-driven for Mb. This entropy-driven feature for P-450_cam is enhanced by the dissociating d-camphor. The on and off activation processes: The on and off reactions for P-450_cam.are dominantly controlled by the enthalpy and entropy terms, respectively, while those for Mb are rather the reverse of the case of P-450_cam. The dissociation of d-camphor has a significant effect on the on reaction but no effect on the off reaction. Analyzing these thermodynamic features on the basis of the physical chemistry in the solution reaction, it was found that these characteristic profiles arise from the difference in the global structural change between the proteins. Namely, during the equilibrium process of the CO binding, this structural change is accompanied by a larger increase in the degree of freedom in P-450_cam than in Mb. We discussed the correlations between the structural changes and their biological significance.     

Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones

The stereoselective reduction of ketones catalysed by alcohol dehydrogenase from Thermoanaerobium brockii was studied in different reaction media, hexane at controlled water activities, hexane with 2. 5% water (biphasic) and pure water. The reactions were studied in the temperature range from -1 to 50 degrees C. Increasing the water activity from 0.53 to 0.97 increased the reaction rate 16-fold. The rate was further enhanced in hexane when exceeding the water solubility and in pure water the rates were even higher. This was general for all ketones studied. At controlled water activity the entropy of activation (DeltaSdouble dagger) was the dominating factor. Large negative DeltaSdouble dagger values caused low reaction rates at low aw. When increasing the carbon chain length of the substrate, for reactions in hexane, the decrease of reaction rate was mainly due to a decrease in DeltaSdouble dagger. In the comparison between hexane and pure water, DeltaGdouble dagger values were higher in hexane due to higher DeltaHdouble dagger (activation enthalpy) values. The enantioselectivity (E value) increased from 2.6 at water activity 0. 53 to 4.6 at water activity 0.97. Changing media from hexane (2.5%, v/v water) to pure water was not affecting the enantioselectivity or the specificity for different ketones.     

Thermodynamic aspects of the gelatinization and swelling of crosslinked starch

Samples of epichlorohydrin crosslinked potato starch were prepared by using a high ratio of starch to water and alkali concentration below the gelatinization level. This yielded crosslinked samples that were partially crystalline, and where the number of crosslinks could be varied between 1 and 20 crosslinks per 100 anhydroglucose units. The degree of swelling varied regularly with degree of crosslinking, and the molecular weight between crosslinks M_c as derived from swelling data in a good swelling agent compared favorably with M_c derived from chemical analysis. From the heat of gelatinization of the crosslinked starches, as observed in a differential scanning calorimeter, for gelatinization in glycerol, water, and dimethylsulfoxide, a model for the gel state of the crosslinked starch is proposed. It is concluded that the entropy of melting is the determining factor in establishing the temperature of gelatinization.