The effect of temperature on the self-association of S5 and on the association of S5 with S8 as determined by sedimentation equilibrium

Solutions of proteins S5 and S8 from the Escherichia coli 30 S ribosomal subunit have been examined by sedimentation equilibrium methods as a function of temperature for their behavior in solution as isolated components and in mixtures. The standard enthalpy and entropy at 4 °C for the isodesmic self-association of S5 were determined from a study over the temperature range of 3 to 33 °C to be 0.1 ± 0.9 kcal/mol and 18 ± 3 cal/(mol × deg), respectively. The protein S8 remained monomeric over the same range of temperature. The standard enthalpy and entropy at 4 °C for the association of S5 and S8 were determined on mixtures from a study over the temperature range of 3 to 27 °C to be ?0.4 ± 1.6 kcal/mol and 20 ± 6 cal/(mol × deg), respectively. Based on these values and the previously determined standard Gibbs free energies (S. H. Tindall and K. C. Aune, 1981, Biochemistry20, 4861–4866), the driving force for the self-association of S5 and the association of S5 with S8 could be interpreted as being derived from the expulsion of water upon ion pair formation at the interaction sites.     

Relationship between the Electrical and Rheological Properties of Potato Tuber Tissue after Various Forms of Processing

The impedance at frequencies of 1-1000 kHz and dynamic bending storage modulus measured by the vibrating reed method were compared for potato tuber tissue, which had been processed by various methods. Raw potato tuber tissue strips were either heated for 30 min up to 100°C or frozen-thawed. Some samples were osmotically dehydrated in a mannitol solution up to a concentration of 0.7 mol/l. The electrical reactance correlated well with the storage modulus of heated or frozen-thawed potato tissues, but not with the storage modulus of the mannitol-treated tissue. The storage modulus appeared to be strongly dependent on the turgor pressure of the cells which was drastically decreased by the heating, freezing-thawing, and osmotic treatments. The electrical properties reflect the cell integrity, and a large difference was observed between the change in impedance after heating or freezing-thawing, and that after the osmotic treatment. A significant change in the electric properties was also observed for a starch suspension at the gelatinization temperature. However, the contribution due to gelatinization did not appear to play an important role in the change of electrical properties of potato tissue by heating.     

Reversible thermal unfolding of thermostable phosphoglycerate kinase. Thermostability associated with mean zero enthalpy change.

Reversible thermal denaturation of phosphoglycerate kinases (E.C. 2.7.2.3) from an extremely thermophilic bacterium Thermus thermophilus and from yeast were studied by measuring their circular dichroism and fluorescence intensity. The thermal denaturation in the presence of guanidine hydrochloride was completely reversible. The thermodynamic parameters for the reaction were calculated based on a two-state mechanism. The free energy changes in denaturation at 25 °C in the absence of denaturant were estimated to be 11.87 ± 0.21 kcal/mol for T. thermophilus phosphoglycerate kinase and 5.33 ± 0.13 kcal/mol for that of yeast. It was found that the van't Hoff plot of the equilibrium constant for the denaturation reaction was almost independent of temperature in the temperature range 0 to 60 °C for T. thermophilus phosphoglycerate kinase, while that of yeast phosphoglycerate kinase was strongly temperature-dependent as reported for other thermolabile proteins. The enthalpy change in denaturation varies from 0.03 to 6.2 kcal/mol (0 to 60 °C) for T. thermophilus phosphoglycerate kinase and from ?27 to 31 kcal/mol (10 to 35 °C) for yeast enzyme. The entropy change in denaturation varies from ?3.9 to 21 entropy units for T. thermophilus phosphoglycerate kinase and ?96 to 104 entropys unit (10 to 35 °C) for yeast enzyme. The heat capacity change in denaturation is between 1.4 and 63 cal/deg. mol for the thermophile enzyme and between 1530 and 1750 cal/deg. mol for yeast enzyme at 20 °C. The observations that the enthalpy changes as well as the heat capacity changes in denaturation of the thermophilic enzyme were negligibly small suggest an explanation for the unusual stability to heat of T. thermophilus phosphoglycerate kinase.We also propose three possible mechanisms for the thermostability of proteins in general.     

Gelatinization mechanism of potato starch

The non-Newtonian behavior and dynamic viscoelasticity of potato starch (Jaga kids red ’90, 21.0% amylose content) solutions after storage at 25 and 4°C for 24 h were measured with a rheogoniometer. The flow curves, at 25°C, of potato starch showed plastic behavior >1.0% (w/v) after heating at 100°C for 30 min. A gelatinization of potato starch occurred above 1.0% at room temperature. A very large dynamic viscoelasticity was observed when potato starch solution (3.0%) was stored at 4°C for 24 h and stayed at a constant value with increasing temperature. A small dynamic modulus of potato starch was observed upon addition of urea (4.0 M) at low temperature (0°C) even after storage at 25 and 4°C for 24 h. A small dynamic modulus was also observed in 0.05 M NaOH solution. Possible models of gelatinization and retrogradation mechanism of potato starch were proposed.     

Interaction of 3’-O-caffeoyl D-quinic acid with multisubunit protein helianthinin

Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present inHelianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6.0. The protein-polyphenol complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined by equilibrium dialysis, at 31°C has a value of 3.5 ± 0.1 × 10⁴M^−-1 resulting in a ΔG value of − 6.32 ± 0.12 kcal /mol. The association constantK _ais 1.0 ± 0.1 × 10⁴M⁻¹ as determined by ultraviolet difference spectral titration at 25°C with ΔG° of -5.46 ± 0.06 kcal/mol. From fluorescence spectral titration at 28°C, theK _avalue is 1.38 ± 0.1 × 1 0 4M⁻¹ resulting in a ΔG of − 5.70 ± 0.05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of binding. The thermal denaturation temperature,T _mof the protein decreases from 76°C to 72°C at 1 × 10⁻³M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect of temperature onK _aof chlorogenic acid shows a nonlinear increase from 10.2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit protein, helianthinin. Publication No. 324.     

Starch Granule Hydration—A MAS NMR Investigation

Starch is the most important energy resource in human diet, and starch is used extensively as a food ingredient to manipulate the quality of our food. In both applications, starch functionality is intimately related to its hydration level. This paper aims at elucidating the starch granule hydration by investigating genotype-specific differences for native wheat, maize, and potato starches by ¹H high-resolution (HR) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. The preparations as analyzed in D₂O suspensions at room temperature provided NMR spectra with large differences in signal-to-noise (S/N) ratio ranging over several orders of magnitude. It was possible to assign a wide range of components including anomeric α-1,4 and α-1,6-protons from reducing and non-reducing ends, respectively. We utilized the effect that only mobile protons (e.g, dissolved or partially hydrated) are observed using ¹H HR-MAS spectroscopy, whereas immobile protons (e.g., in water-inaccessible regions) of the starch granule are not observed due to strong homonuclear interactions to verify the hypothesis that the variations in signal intensities between the different starches are caused by genotype-specific variations in assembly of the starch granules and that the signal intensity, thus, indicates the extent of accessible granule hydration surfaces. Moreover, events taking place during thermal starch granule hydration (gelatinization) were investigated for ten representative starches. NMR spectra of suspended samples were acquired at 30, 45 and 70 °C and again after cooling at 30 °C. A substantial increase in NMR signal intensity occurs above the gelatinization temperature due to extensive proton mobilization in the starch granule assembly. The relative integrated spectral intensities at 30 °C before and after gelatinization at 70 °C showed differences in gain factors between 4 and 193. Also, ³¹P MAS NMR spectra displayed a similar significant intensity gain upon gelatinization. The results showed that the phosphate groups in the starch granule are mobilized concomitantly with the protons and thus deeply “buried” in the immobile (water inaccessible) domains.     

Thermodynamics of the slow–fast transition in bacteriophage T2L

We have measured the thermodynamic parameters of the slow-fast tail-fiber reorientation transition on T2L bateriophage. Proportions of the virus in each form were determined from peak-height measurements in sedimention-velocity runs and from average diffusion coefficients obtained by quasielastic laser light scattering. Computer simulation of sedimentation confirmed that there were no undetected intermediates in the transition, which was analyzed as a two-state process. Van't Hoff-type plots of the apparent equilibrium constant and of the pH midpoint of the transition as function of reciprocal temperature led to the following estimates of the thermodynamic parameters for the transition at pH 6.0 and 20°C: ΔH° = ?139 ± 18Kcal mol^?1, ΔS° = ?247 ± 46 cal K^?1 mol^?1, and ΔG° = ?66 ± 22 kcal mol^?1. Per mole of protons taken up in the transition, the analogous quantities were ?15.9 ± 1.7 kcal mol^?1, ?26.3 ± 2.2 cal K^?1 mol^?1, and ?8.22 ± 1.8 kcal mol^?1. The net number of protons taken up was about 8.5 ± 1.5. The large values of the thermodynamic functions are consistent with a highly cooperative reaction and with multiple interactions between the fibres and the remainder of the phage. The negative entropy of the transition is probably due to immobilization of the fibres.     

High alcohol production by solid substrate fermentation from starchy substrates using thermotolerant Saccharomyces cerevisiae

Solid Substrate Fermentation system (SSF) was used to produce ethanol from various starchy substrates like sweet sorghum, sweet potato, wheat flour, rice starch, soluble starch and potato starch using thermotolerant yeast isolate (VS₃) by simultaneous saccharification and fermentation process. Alcohol produced was estimated by gas chromatography after an incubation time of 96 hrs at 37v°C and 42v°C. More ethanol was produced from rice starch and sweet sorghum. The maximum amount of ethanol produced from these substrates using VS₃ was 10 g/100 g and 3.5 g/100 g substrate (rice starch) and 8.2 g and 7.5 g/100 g substrate (sweet sorghum) at 37v°C and 42v°C respectively.     

The interaction of albumin and concanavalin A with normal and sickle human erythrocytes.

The interaction of human albumin and concanavalin A with normal and sickle human red blood cells previously washed in phosphate buffer at pH = 7.4 was studied by titration calorimetry. The amount of albumin bound to normal cells was (6.8 ± 2.2) × 10⁵ molecules/cell. An equilibrium constant of 5 × 10¹⁰ and an enthalpy change of ?(280 ± 90) kcal/mol albumin was determined for albumin interaction with normal cells. The amount of albumin bound to sickle cells was (12.4 ± 1.0) × 10⁵ molecules/cell and the enthalpy change for albumin interaction with sickle cells was ?(390 ± 140) kcal/mol. Normal cells bound (5.7 ± 2.4) × 10⁵ concanavalin A molecules/cell with an enthalpy change of ?(840 ± 200) kcal/mol concanavalin. All experiments were conducted at 25°C.     

Temperature-jump kinetics of the dC-G-T-G-A-A-T-T-C-G-C-G double helix containing a G . T base pair and the dC-G-C-A-G-A-A-T-T-C-G-C-G double helix containing an extra adenine

The kinetics of helix formation were investigated using the temperature-jump technique for the following two molecules: dC-G-T-G-A-A-T-T-C-G-C-G, which forms a double helix containing a G·T base pair(the G·T 12-mer), and dC-G-C-A-G-A-A-T-T-C-G-C-G, which forms a double helix containing an extra adenine (the 13-mer). When data were analyzed in an all-or-none model, the activation energy for the helix association process was 22 ± 4 kcal/mol for the G·T 12-mer and 16 ± 7 kcal/mol for the 13-mer. The activation energy for the helix-dissociation process was 68 ± 2 kcal/mol for the G·T 12-mer and 74 ± 3 kcal/mol for the 13-mer. Rate constants for recombination were near 10⁵s^?1M^?1 in the temperature range from 32 to 47°C; for the dissociation process, the rate constants varied from 1s^?1 near 32°C to 130s^?1 near 47°C. Possible effects of hairpin loops and fraying ends on the above data are discussed.     

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca²⁺. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (T_M) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔH_c) of 105 ± 15 kcal/mol, in the absence of Ca²⁺. In the presence of Ca²⁺ concentrations sufficient to saturate its sites on Factor IX, the T_m value is increased to 57.0 ± 0.5 °C and the ΔH_c is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca²⁺, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca²⁺, Factor IXα displayed thermograms characterized by a T_M of 51.0 ± 0.5 °C and a ΔH_c of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca²⁺ (the values in the presence of saturating Ca²⁺ are given in parentheses), undergoes thermal denaturation with a T_M of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔH_c of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a T_M of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔH_c of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a T_M of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a T_M of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca²⁺ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.     

Purification and characterization of α-amylase fromAspergillus flavus

Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate— polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5±2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55°C and it was stable for 1 h up to 50°C. TheK _m andV for gelatinized tapioca starch were 0.5 g/L and 108.67 μmol reducing sugars per mg protein per min, respectively.     

Helix-coil dynamics of a Z-helix hairpin

The helix–coil transition of a Z-helix hairpin formed from d(C-G)₅T₄(C-G)₅ has been characterized by equilibrium melting and temperature jump experiments in 5M NaClO₄ and 10 mM Na₂HPO₄, pH 7.0. The melting curve can be represented by a simple all-or-none transition with a midpoint at 81.6 ± 0.4°C and an enthalpy change of 287 ± 15 kJ/mole. The temperature jump relaxation can be described by single exponentials at a reasonable accuracy. Amplitudes measured as a function of temperature provide equilibrium parameters consistent with those derived from equilibrium melting curves. The rate constants of Z-helix formation are found in the range from 1800 s^?1 at 70°C to 800 s^?1 at 90°C and are associated with an activation enthalpy of ?(50 ± 10) kJ/mole, whereas the rate constants of helix dissociation are found in the range from 200 s^?1 at 70°C to 4500 s^?1 at 90°C with an activation enthalpy +235 kJ/mole. These parameters are consistent with a requirement of 3–4 base pairs for helix nucleation. Apparently nucleation occurs in the Z-helix conformation, because a separate slow step corresponding to a B to Z transition has not been observed. In summary, the dynamics of the Z-helix–coil transition is very similar to that of previously investigated right-handed double helices.     

Preparation of Triglyceride Hydroperoxides

Corticium rolfsii AHU 9627, which we isolated from a tomato stem, is one of the most promising producers of a raw starch saccharifying enzyme. The effects of the cultural conditions and medium components on the enzyme production were investigated. The enzyme production was improved by increasing both the concentrations of carbon sources and organic nutrients in the medium. Under the optimum cultural conditions, the enzyme activity of the culture supernatant against raw starch reached a maximum after 8-days incubation at 27°C and the activity reached 80 units per ml (when determined at 40°C and pH 4.0). The optimal pH and temperature for the enzyme reaction were 4.0 and 65°C, respectively. The saccharifying reaction was scarcely inhibited even with a high substrate concentration, and raw starch was rapidly hydrolyzed into glucose.     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

The effect of different patterns of thermal processing of potatoes on their digestibility by growing pigs

The effects of three patterns of thermal processing of potatoes on the sites and efficiency of their digestion were examined using pigs cannulated at the terminal ileum. The potatoes were processed in each of the following ways: (I) steamed for 1 h at 100°C, slowly cooled during 24 h to 40°C and then rapidly equilibrated with ambient temperature; (II) steamed for 20 min at 100°C, then rapidly cooled; (III) steamed at 70°C for 20 min, then rapidly cooled. They were given with a mineral and vitamin supplement in three meals per day and the allowance of feed was 70 g of organic matter/kg W^0.75 (～ 9.6 kg “fresh” potato per pig/day). Ileal digesta and faeces were collected during 12-h periods, sub-divided for ileal samples into three periods of 4 h, each commencing after a meal.The results indicate that Treatment I was undesirable; the prolonged heating caused rupture of the cell walls with release of gelatinized starch and resulted in potato protein of low digestibility, particularly anterior to the ileo-caecal junction. Treatment II gave the best results. Treatment III did not cause complete gelatinization of the potato starch and did not effectively denature the anti-nutritive protein present in the raw potato, contrary to the results of laboratory studies. Coefficients of digestibility of organic matter and nitrogen, measured at the terminal ileum, were reduced by 13 and 48% compared with those obtained in Treatment II. Corresponding reductions over the whole gut were 3 and 19%, respectively.     

The standard Gibbs free energy change of hydrolysis of alpha-D-ribose 1-phosphate

The standard Gibbs free energy change of hydrolysis of α-d-ribose 1-phosphate has been measured at pH 7.0, ionic strength 0.1 m, and 25 °C by combining the corresponding values of the two following reactions: adenosine + H₂O ág adenine + ribose (ΔG^0′ = ?2.3 ± 0.1 kcal/mol), catalyzed by adenosine nucleosidase, and ribose 1-phosphate + adenine ág adenosine + P_i (ΔG^0′ = ?3.1 ± 0.1 kcal/mol), catalyzed by adenosine phosphorylase. The standard Gibbs free energy changes were calculated for both reactions from the equilibrium constant. A value of -5.4 ± 0.15 kcal/mol, comparable to that of other hemiacetal phosphoric esters, was obtained for the hydrolysis of ribose 1-phosphate.     

Purification and properties of Bacillus coagulans cyclomaltodextrin glucanotransferase

Cyclomaltodextrin glucanotransferase (CGTase), produced in a culture filtrate by Bacillus coagulans, was purified to a single, homogeneous protein. It has a monomeric structure with a molecular weight of 65,000, isoelectric point of 4.6, and contains 2 mol of Ca²⁺ per mol of the enzyme. The enzyme was most active at pH 6.0 and at 70°C. It did not lose its activity by heat treatment at 70°C for 10 min in the presence of CaCl₂ in the pH range of 5.5∼9.5, and by incubation in the pH range of 5.0∼10.5 at 4°C for one month. The enzyme converted about 60% of potato starch to cyclodextrins for 20 h at 50°C, and the ratio of α-: β-: γ-cyclodextrin produced was 8.1:8.9:1.0 B. coagulans CGTase was compared with B. macerans CGTase which was purified by the same method.     

Influence of pH and ionic strength on the viscoelastic properties of starch gels — A comparison of potato and cassava starches

The influence of pH, and of electrolytes, on the viscoelastic properties of potato and cassava starch gels was investigated, using a cone-and-plate rheometer run in the oscillatory mode. The gel strength of the potato starch gels had a maximum around pH 8·5, and was markedly lowered by the addition of even small amounts of electrolytes. This may be due to an electrostatic interaction between potato starch phosphate groups and added cations which blocks the normal phosphate-to-phosphate cross-linking. Neither pH nor electrolytes affected the viscoelastic properties of cassava starch gels. The gelatinization temperature and the gelatinization enthalpy of potato starch, as measured by differential scanning calorimetry, were insensitive to pH and to low electrolyte concentrations.