Commercial baker's yeast stability as affected by intracellular content of trehalose, dehydration procedure and the physical properties of external matrices

The effects of vacuum-drying and freeze- drying on the cell viability of a commercial baker's yeast, Saccharomyces cerevisiae, strain with different endogenous contents of trehalose were analyzed. An osmotolerant Zygosaccharomyces rouxii strain was used for comparative purposes. Higher viability values were observed in cells after vacuum-drying than after freeze-drying. Internal concentrations of trehalose in the range 10–20% protected cells in both dehydration processes. Endogenous trehalose concentrations did not affect the water sorption isotherm nor the T _g values. The effect of external matrices of trehalose and maltodextrin was also studied. The addition of external trehalose improved the survival of S. cerevisiae cells containing 5% internal trehalose during dehydration. Maltodextrin (1.8 kDa) failed to protect vacuum-dried samples at 40 °C. The major reduction in the viability during the freeze-drying process of the sensitive yeast cells studied was attributed to the freezing step. The suggested protective mechanisms for each particular system are vitrification and the specific interactions of trehalose with membranes and/or proteins. The failure of maltodextrins to protect cells was attributed to the fact that none of the suggested mechanisms of protection could operate in these systems. Received: 6 December 1999 / Received revision: 8 May 2000 / Accepted: 19 May 2000     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.     

Effect of xylitol and trehalose on dry resistance of yeasts

The effects of dehydration/rehydration on two strains of Saccharomyces cerevisiae: S600, a metabolically engineered xylose-utilising strain, and H158, the non-xylose-utilising host strain; and on the naturally xylose-utilising yeast Pachysolen tannophilus CBS 4044, were compared after glucose and xylose utilisation respectively. The yeast strains differed in their ability to excrete and accumulate intracellular xylitol. A high intracellular xylitol content before and after dehydration coincided with a higher viability after a dehydration/rehydration cycle. The intracellular trehalose content increased during dehydration in all three yeast strains, but this did not correspond to enhanced cell viability after dehydration/rehydration. The results are discussed in relation to the ability of xylitol and trehalose to structure water. Received: 9 July 1996 / Received revision: 29 October 1996 / Accepted: 2 November 1996     

The State of Aggregation of Casein Affects the Storage Stability of Amorphous Sucrose, Lactose, and Their Mixtures

The effect of the state of aggregation of casein (micellar or non-micellar, as milk protein concentrate [MPC] or sodium caseinate [Na-caseinate], respectively) on water sorption, plasticization, and crystallization of freeze-dried matrices containing sucrose, lactose or their blends were studied. The Guggenheim–Anderson–de Boer (GAB) equation satisfactorily fitted to the water sorption data. In most cases, sugar crystallization—studied by water sorption behavior, x-ray diffraction, and non-isothermal calorimetry—occurred significantly slower in systems containing Na-caseinate compared to MPC. The type of casein did not affect the temperature range where the glass transition (T _g) was observed. Sugar/Na-caseinate mixtures showed higher instant crystallization temperatures (up to 20°C) than sugar/MPC mixtures. X-ray diffraction showed that: (a) crystallinity increased with increasing relative vapor pressure (RVP) > 44%; (b) lactose crystallized mainly as α-lactose monohydrate regardless of casein type; and (c) that sucrose crystals predominated the patterns of the sucrose/lactose mixtures. Results suggested that the way proteins organize in space (i.e., aggregation state) affected their interactions with neighboring sugar and water molecules, which led to differences in sugar crystallization behavior. Poster presented at the 4th International Workshop on Water in Food in Brussels March, 2006. Funded by CONACyT (Mexico) and Dippin’ Dots Inc., KY, USA.     

Understanding the Influence of State/Phase Transitions on Ice Recrystallization in Atlantic Salmon (Salmo salar) During Frozen Storage

Temperature fluctuations during storage and distribution of frozen foods lead to ice recrystallization and microstructural modifications that can affect food quality. Low temperature transitions may occur in frozen foods due to temperature fluctuations, resulting in less viscous and partially melted food matrices. This study systematically investigated the influence of state/phase transitions and temperature fluctuations on ice recrystallization during the frozen storage of salmon fillets. Using a modulated differential scanning calorimeter, we identified the characteristics glass transition temperature (T _g ′) of −27 °C and the onset temperature for ice crystal melting (T _m ′) of −17 °C in salmon. The temperature of salmon fillets in sealed plastic trays was lowered to −35 °C in a freezer to achieve the glassy state. The temperature (T) of frozen salmon fillets in sealed plastic trays was modulated to achieve a rubbery state (T > T _m ′), a partially freeze-concentrated state (T _g ′ < T < T _m ′) and a glassy state (T < T _g ′). We performed temperature modulation experiments by exposing packaged salmon to room temperature twice a day for 2 to 26 min during 4 weeks of storage. We also analyzed ice crystal morphology using environmental scanning electron microscopy and X-ray computed tomography techniques to observe the pore distribution after sublimation of ice crystals. Melt–refreeze and isomass rounding mechanisms of ice recrystallization were noticed in the frozen salmon subjected to temperature modulations. Results show that ice crystal growth occurred even in the glassy state of frozen salmon during storage, with or without temperature fluctuations. Ice crystal size in frozen salmon was greater in the rubbery state (T > T _m ′) due to the increased mobility of unfrozen water compared to the glassy state. The morphological/geometric parameters of ice crystals in frozen salmon stored for 1 month differed significantly from those in 0-day storage. These findings are important to the frozen food industry because they can help optimize storage and distribution conditions and minimize quality loss of frozen salmon due to recrystallization.     

Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag at the NH₂-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis. Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998     

Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger

The Aspergillus niger strain BO-1 was grown in batch, continuous (chemostat) and fed-batch cultivations in order to study the production of the extracellular enzyme glucoamylase under different growth conditions. In the pH range 2.5–6.0, the specific glucoamylase productivity and the specific growth rate of the fungus were independent of pH when grown in batch cultivations. The specific glucoamylase producivity increased linearly with the specific growth rate in the range 0–0.1 h⁻¹ and was constant in the range 0.1–0.2 h⁻¹. Maltose and maltodextrin were non-inducing carbon sources compared to glucose, and the maximum specific growth rate was 0.19 ± 0.02 h⁻¹ irrespective of whether glucose or maltose was the carbon source. In fed-batch cultivations, glucoamylase titres of up to 6.5 g l⁻¹ were obtained even though the strain contained only one copy of the glaA gene. Received: 5 May 1999 / Received revision: 7 September 1999 / Accepted: 17 September 1999     

Isolation and Identification of γ-l-Glutamyl-l-glutamic Acid from Tobacco Cells in Suspension Culture

The Maillard Reaction (MR) rate below the glass transition temperature (T_g) for various model glassy food systems was studied at temperatures between 40 °C and 70 °C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T_g was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD₂₈₀), and the MR rate (k₂₈₀) was determined as a pseudo zero order reaction rate from the time course of OD₂₈₀. Although k₂₈₀ was described by the Arrhenius plot, the temperature dependence of k₂₈₀ was almost the same and the intercept was different among the matrices. From the comparison of k₂₈₀, it was suggested that the MR rate in glassy matrix was affected not only by the T_g, but also by the hydrogen bonding between MR reactants and glassy matrix.     

Mixed-valent μ-oxo-bridged diiron complexes produced by radiolytic reduction at 77 K studied by EPR

 Several dinuclear diferric complexes were prepared, bridged by only an oxo ion, by an oxo ion and additionally one or two carboxylate groups, or by two hydroxo ions. Frozen solutions of these EPR-silent diferric complexes were radiolytically reduced by γ-irradiation at 77 K. The reduced forms gave EPR spectra typical of mixed-valent diiron species with S = 1/2. The yield was about 50% at high doses. With ⁵⁷Fe replacing ⁵⁶Fe the spectral features were broadened because of hyperfine interaction with one or both of the ⁵⁷Fe nuclei. All oxo-bridged complexes give mixed-valent species with unbroadened EPR spectra observable up to 110 K. The anisotropy is small, Δg = g ₁–g ₃<0.07, and g _av = (g ₁+g ₂+g ₃)/3 falls in the range 1.93–1.96. Annealing at T≥115 K causes the EPR spectra to change and finally become more anisotropic, 0.14<Δg<0.32 and to have smaller g _av, 1.78<g _av<1.89. These spectra are observable only at T≤35 K. Radiolytic reduction of the diferric complexes with hydroxo bridges leads directly without annealing to this second type of mixed-valent species. The annealing-induced transition from a primary to a secondary mixed-valent species in the case of an oxo-bridged diferric starting complex is suggested to be a structural relaxation including protonation of the bridge. Annealing at temperatures above ca. 180 K and final thawing causes the mixed-valent dinuclear species to disappear, probably by disintegration to mononuclear species. These mixed-valent species are useful models for similar species in proteins, where primary-type species may take part in transient electron transfer, and the secondary-type species are usually more persistent forms in redox reactions. For proteins, available data show that there is a tendency for both primary and secondary species to have larger g anisotropy, Δg, and smaller g _av, than obtained with the models. These differences suggest that the diiron centers of proteins are affected by their specific surroundings. Our results also show that the nature of the bridge, oxo or hydroxo, may not always safely be determined from a measured J value. Measurement of the EPR parameters, Δg, g _av and limiting temperature of observation (relaxation properties) of a mixed-valent complex, or, in the case of a diferric complex, of the primary mixed-valent species induced by radiolysis at 77 K, offers an alternative empirically based method to determine the nature of the bridge. Received: 8 May 1996 / Accepted: 22 January 1997     

Production of poly-(β-hydroxybutyrate) from saponified Vernonia galamensis oil by Alcaligenes eutrophus

Saponified vernonia oil was converted exclusively to poly(β-hydroxybutyrate) (PHB) by Alcaligenes eutrophus in a single-stage batch culture. After harvesting, centrifugation followed by lyophilization, the resulting dried cells contained up to 42.8 wt% PHB having a peak molecular mass of 381 863 Da, weight-average molecular mass of 308 390 Da, and a polydispersity of 1.1. The PHB had a melting point (T_m) range of 163–174°C with a maximum at 172°C (lit. T_m, 175°C), and heat of fusion of 18.43 cal g⁻¹. Fermentation performed under varying conditions of nitrogen limitation indicated that there was no significant effect of nitrogen concentration on the molecular mass of PHB produced from vernonia oil by A. eutrophus. Received 27 March 1998/ Accepted in revised form 17 July 1998     

The Effect of Molecular Size on Molecular Mobility in Amorphous Oligosaccharides

The physical properties and especially the molecular mobility of amorphous carbohydrate matrixes directly influence the stability of foods, feeds, and pharmaceuticals and the dessication tolerance of animals and plants during anhydrobiosis. Phosphorescence of the sodium salt of erythrosin B was used to investigate the local molecular mobility in pure amorphous solids of a homologous series of malto-oligosaccharides (maltose, G₂; maltotriose, G₃; maltotetraose, G₄; maltopentaose, G₅; maltohexaose, G₆; and maltoheptaose, G₇); sucrose and maltodextrin DE18 (a hydrolytic fraction of starch) were investigated for comparison. Measurements of the temperature-dependence of the phosphorescence emission energy, an indicator of the extent of local dipolar relaxation, and the phosphorescence emission lifetime, an indicator of the rate of collisional quenching of the excited state by matrix molecules, demonstrate that the local matrix molecular mobility increases with molecular size, and thus, with an increase in T _g, in these glucose oligosaccharides. Master curves of the spectroscopic measures of matrix mobility for each oligosaccharide, plotted against T–T _g, were not superimposable, suggesting that local properties of the amorphous sugar matrixes, rather than T _g per se, influence local matrix mobility. Indicators of spectral heterogeneity also varied with molecular size, indicating that dynamic site heterogeneity also increased with molecular size and thus, T _g. These results emphasize the importance of additional research in developing appropriate “molecular rules” for designing amorphous matrix systems with better long-term stability for foods, feeds, or pharmaceuticals.     

Trehalose–Water–Salt Interactions Related to the Stability of β-Galactosidase in Supercooled Media

The conservation of desirable properties in foods and ingredients is often based on the maintenance of the amorphous metastable properties of the systems. Enzymes may be stabilized by drying in saccharide matrices, but a second excipient is generally required to improve sugar protective effects. The effect of electrolytes on the thermophysical properties of sugar systems is of special interest because of their major influence on water structure and their possible interactions with biomolecules. Salts affect the kinetics of very important changes in sugar systems such as crystallization. The purpose of the present work was to analyze fungal β-galactosidase stability in supercooled systems of trehalose-containing electrolytes (water soluble acetates, citrates, and chlorides of magnesium and potassium). The degree of sugar crystallization was also related to enzyme stability. Potassium citrate and acetate improved enzyme stability during freeze-drying and thermal treatment of samples at water activity (a _w) of 0.22. However, trehalose crystallization inhibition at a _w = 0.43 (which was about 50–60%, related to the system without salt) impaired enzyme protection. Certain salts may act retarding sugar crystallization, but in the presence of salts, trehalose crystallization is even more critical because the enzyme is confined in a highly salt-concentrated region. Thus, crystallization inhibition by sugar–salt combinations should be carefully conducted. Santagapita, Research Fellow, CONICET, Argentina. Buera, Member of CONICET, Argentina. An erratum to this article can be found at     

Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp

Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers). Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released (K _v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from Penicillium simplicissimum (K _v = 0.15 l mPa⁻¹s⁻¹g⁻¹) and a mannanase from Sclerotium rolfsii (K _v = 0.12 l mPa⁻¹s⁻¹g⁻¹) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO respectively. A less significant brightness increase was obtained with enzymes showing lower K _v values, such as a xylanase from Schizophyllum commune (K_v = 0.051  l mPa⁻¹s⁻¹g⁻¹, 0.2% ISO) and a bacterial mannanase (K _v = 0.061 l mPa⁻¹s⁻¹g⁻¹,0.5% ISO). Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997     

Purification, characterization, and immunolocalization of hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase from opium poppy

A development-specific and elicitor-inducible acyltransferase [hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110)] that catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to hydroxyphenethylamines was purified 988-fold to apparent homogeneity from opium poppy (Papaver somniferum L.) cell-suspension cultures. The purification procedure, which resulted in a 6.8% yield, involved hydrophobic interaction and anion-exchange chromatography, followed by affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent. Purified THT had an isoelectric point of 5.2, a native molecular mass of approximately 50 kDa, and consisted of two apparently identical 25-kDa subunits as determined by two-dimensional polyacrylamide gel electrophoresis. The purified enzyme was able to synthesize a variety of amides due to a relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines. The best substrates were feruloyl-CoA (VK _m ⁻¹13.4 mkat g⁻¹ M⁻¹) and tyramine (VK _m ⁻¹6.57 mkat g⁻¹ M⁻¹). The THT activity increased during development of opium poppy seedlings, occurred at high levels in roots and stems of mature plants, and was induced in cell-suspension cultures after treatment with a pathogen-derived elicitor. Immunoblot analysis using THT mouse polyclonal antibodies did not always show a correlation between THT polypeptide and enzyme activity levels. For example, despite low THT activity in leaves, an abundant 25-kDa immunoreactive polypeptide was detected. Immunohistochemical localization showed that THT polypeptides occur in cortical and xylem parenchyma, immature xylem vessel elements, root periderm, anthers, ovules, and the inner layer of the seed coat, but are most abundant in phloem sieve-tube members in roots, stems, leaves, and anther filaments. Received: 19 January 1999 / Accepted: 3 March 1999     

Use of alginate and cryo-protective sugars to improve the viability of lactic acid bacteria after freezing and freeze-drying

Summary In the present paper, the effect of cryo-protective sugars on the survival rate of different strains of Lactic Acid Bacteria (LAB, Lactobacillus acidophilus, Lactobacillus delbrueckii subspbulgaricus, Streptococcus salivarius subsp.thermophilus), after freezing or freeze-drying procedures, was compared. The cells were incubated at 4 °C in 32% final concentration sugar solutions (trehalose, maltose, sucrose, glucose and lactose), and viability was evaluated by the enumeration of colony-forming units. All sugars tested showed a protective effect on cell viability as compared to isotonic solution, especially after freeze-drying procedures (log c.f.u./ml ranging between 1.16 and 2.08, P < 0.001). Furthermore, the resistance to different stress agents (lysozyme, pepsin, bile salts) was estimated. Trehalose was the most effective sugar in preserving bacterial viability [% (log c.f.u. trehalose/log c.f.u. isotonic solution) ranging between 124 and 175, P < 0.001] although each strain showed a different sensitivity. Finally, the protective effect of immobilization of LAB in Ca-alginate beads was compared to that exercised by trehalose. The immobilization induced a good survival rate but lower as compared to the trehalose effect, mainly after freeze-drying in the presence of the selective agents [% (log c.f.u. alginate/log c.f.u. trehalose ranging between 81.1 and 94.5, P < 0.0001]. The protective effect of trehalose was evident in particular for Lactobacillus delbrueckii subsp. bulgaricus in presence of lysozyme. Therefore, because of its chemical inertness and low cost, trehalose could be easily utilized as excellent bacterial preservative, both to improve the viability of starter cultures and to obtain probiotic formulations more resistant to a variety of stressful conditions.     

A combined experimental and computational study on the material properties of shape memory polyurethane

A type of shape memory polyurethane with 60 wt% hard segments (SMPU60) was prepared. Its material properties were tested by dynamic mechanical analysis (DMA) and Instron, and simulated using fully atomistic molecular dynamics (MD). The glass transition temperature (T _g) of SMPU60 determined by DMA is 316 K, which is slightly lower than that estimated through MD simulations (T _g = 328 K) , showing the calculated T _g is in good agreement with experimental data. A complex hydrogen bonding network was revealed with the calculation of radial distribution functions (RDFs). The C═O⋯H bond is the predominant hydrogen-bonding interaction. With increasing temperature, both the hydrogen bonding and the moduli decreased, and the dissociation of intermolecular hydrogen bonding induced the decrease of the moduli.     

The production of xylitol from d-xylose by fermentation with Hansenula polymorpha

We have analysed the influence of the initial pH of the medium and the quantity of aeration provided during the batch fermentation of solutions of d-xylose by the yeast Hansenula polymorpha (34438 ATCC). The initial pH was altered between 3.5 and 6.5 whilst aeration varied between 0.0 and 0.3 vvm. The temperature was kept at 30 °C during all the experiments. Hansenula polymorpha is known to produce high quantities of xylitol and low quantities of ethanol. The most favourable conditions for the growth of xylitol turned out to be: an initial pH of between 4.5 and 5.5 and the aeration provided by the stirring vortex alone. Thus, at an initial pH of 5.5, the maximum specific production rate (μ_m) was 0.41 h⁻¹, the overall biomass yield (Y _x/s ^G) was 0.12 g g⁻¹, the specific d-xylose-consumption rate (q _s ) was 0.075 g g⁻¹ h⁻¹ (for t = 75 h), the specific xylitol-production rate (q _Xy ) was 0.31 g g⁻¹ h⁻¹ (for t = 30 h) and the overall yields of ethanol (Y _E/s ^G) and xylitol (Y _Xy/s ^G) were 0.017 and 0.61 g g⁻¹ respectively. Both q _s and q _Xy decreased during the course of the experiments once the exponential growth phase had finished. Received: 26 March 1998 / Received revision: 30 June 1998 / Accepted: 2 July 1998     

Thermal ecology of the Australian agamid Pogona barbata

This study compares the thermal ecology of male bearded dragon lizards (Pogona barbata) from south-east Queensland across two seasons: summer (1994–1995) and autumn (1995). Seasonal patterns of body temperature (T _b) were explored in terms of changes in the physical properties of the thermal environment and thermoregulatory effort. To quantify thermoregulatory effort, we compared behavioral and physiological variables recorded for observed lizards with those estimated for a thermoconforming lizard. The study lizards' field T _bs varied seasonally (summer: grand daily mean (GDM) 34.6 ± 0.6°C, autumn: GDM 27.5 ± 0.3°C) as did maximum and minimum available operative temperatures (summer: GDM T _max 42.1 ± 1.7°C, T _min 32.2 ± 1.0°C, autumn: GDM T _max 31.7 ± 1.2°C, T _min 26.4 ± 0.5°C). Interestingly, the range of temperatures that lizards selected in a gradient (selected range) did not change seasonally. However, P. barbata thermoregulated more extensively and more accurately in summer than in autumn; lizards generally displayed behaviors affecting heat load nonrandomly in summer and randomly in autumn, leading to the GDM of the mean deviations of lizards' field T _bs from their selected ranges being only 2.1 ± 0.5°C in summer, compared to 4.4 ± 0.5°C in autumn. This seasonal difference was not a consequence of different heat availability in the two seasons, because the seasonally available ranges of operative temperatures rarely precluded lizards from attaining field T _bs within their selected range, should that have been the goal. Rather, thermal microhabitat distribution and social behavior appear to have had an important influence on seasonal levels of thermoregulatory effort. Received: 28 April 1997 / Accepted: 29 December 1997     

EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999