ON THE QUANTA OF LIGHT PRODUCED AND THE MOLECULES OF OXYGEN UTILIZED DURING CYPRIDINA LUMINESCENCE

A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10^–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10^–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O₂ = oxyluciferin + H₂O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.     

STUDIES ON BIOLUMINESCENCE : XIV. THE SPECIFICITY OF LUCIFERIN AND LUCIFERASE.

Among sixteen groups of luminous forms investigated by the author, in only four (fireflies, Pholas, ostracods, and Odontosyllis) is it possible to demonstrate the luciferin-luciferase reaction. In many groups this is probably due to the small amount of these substances present in the luminescent organism or to their instability. In the medusæ and pennatulids, despite a large amount of luminescent material, luciferin and luciferase cannot be demonstrated. This does not appear to be due to the presence of luciferin and luciferase in equivalent proportion, or to their instability. In fact, one is led to the conclusion that luciferin and luciferase do not exist in these forms, but such a conclusion must be regarded as merely tentative, in view of the fundamental character of the luciferin-luciferase reaction. Luciferin of one form will not luminesce with the luciferase of another form or vice versa, unless very closely related (Cypridina and Pyrocypris). All experiments emphasize the specificity of the light producing substances of Cypridina.     

Effect of pH, Mg, CO(2) and Mercurials on the Circular Dichroism, Thermal Stability and Light Scattering of Ribulose 1,5-Bisphosphate Carboxylases from Alfalfa, Spinach and Tobacco

Circular dichroism, differential scanning calorimetry and light-scattering measurements of ribulose 1,5-bisphosphate carboxylase (E.C. 4.1.1.39) from alfalfa, spinach and tobacco show: a) The conformation and thermal stability of the native carboxylases are sensitive to changes in pH and to activation of the enzyme with Mg²⁺ and CO₂. The helical content, denaturation temperature (T_d) and specific enthalpy of denaturation (Δq) decreased with increase in pH. Addition of Mg²⁺ and CO₂ at pH 9 increased T_d by 4 to 5 C; at pH 7.5 the changes in T_d were smaller. b) Addition of mercurials produced changes in conformation and thermal stability. The decrease in helical content of the enzymes with increase in pH was enhanced by the addition of p-chloromercuribenzoate. At pH 9, addition of p-chloromercuribenzoate or of 1-(3-(chloromercuri)-2-methoxypropyl)urea decreased T_d by 11.4 to 20.2 C and Δq by 2.1 to 2.8 calories per gram. c) The spinach carboxylase undergoes the largest and the tobacco the smallest changes in conformation and thermal stability upon change in pH or treatment with mercurials. d) The calorimetric data suggest that the large and small subunits are heat denatured independently but at the same temperature. e) Light scattering measurements at pH 9 of p-chloromercuribenzoate treated tobacco enzyme showed that there is no dissociation into subunits upon heating to temperatures greater than T_d. A `ball and string' model for the carboxylase molecule is proposed to reconcile independence of subunit denaturation with apparent strong interactions between subunits.     

THE NATURE AND CONTROL OF REACTIONS IN BIOLUMINESCENCE : WITH SPECIAL REFERENCE TO THE MECHANISM OF REVERSIBLE AND IRREVERSIBLE INHIBITIONS BY HYDROGEN AND HYDROXYL IONS,TEMPERATURE, PRESSURE,ALCOHOL, URETHANE,AND SULFANILAMIDE IN BACTERIA

On the basis of available data with regard to the chemical and physical properties of the "substrate" luciferin (LH₂) and enzyme, luciferase (A), and of kinetic data derived both from the reaction in extracts of Cypridina, and from the luminescence of intact bacteria, the fundamental reactions involved in the phenomenon of bioluminescence have been schematized. These reactions provide a satisfactory basis for interpreting the known characteristics of the system, as well as the theoretical chemistry with regard to the control of its over-all velocity in relation to various factors. These factors, here studied experimentally wholly with bacteria, Photobacterium phosphoreum in particular, include pH, temperature, pressure, and the drugs sulfanilamide, urethane, and alcohol, separately and in relation to each other. Under steady state conditions of bacterial luminescence, with excess of oxidizable substrate and with oxygen not limiting, the data indicate that the chief effects of these agents center around the pace setting reactions, which may be designated by the equation: A + LH₂ → ALH₂ following which light emission is assumed proportional to the amount of the excited molecule, AL*. The relation between pH and luminescence intensity varies with (a), the buffer mixture and concentration, (b), the temperature, and (c), the hydrostatic pressure. At an optimum temperature for luminescence of about 22° C. in P. phosphoreum, the effects of increasing or decreasing the hydrogen ion concentration are largely reversible over the range between pH 3.6 and pH 8.8. The relation between luminescence intensity and pH, under the experimental conditions employed, is given by the following equation, in which I ₁ represents the maximum intensity, occurring about pH 6.5; I ₂ the intensity at any other given pH; K ₅ the equilibrium constant between hydrogen ions and the AL_-; and K ₆ the corresponding constant with respect to hydroxyl ions: See PDF for Equation The value of K ₅, as indicated by the data, amounts to 4.84 x 10⁴, while that of K ₆ amounts to 4.8 x 10⁵. Beyond the range between approximately pH 3.8 and 8.8, destructive effects of the hydrogen and hydroxyl ions, respectively, were increasingly apparent. By raising the temperature above the optimum, the destructive effects were apparent at all pH, and the intensity of the luminescence diminished logarithmically with time. With respect to pH, the rate of destruction of the light-emitting system at temperatures above the optimum was slowest between pH 6.5 and 7.0, and increased rapidly with more acid or more alkaline reactions of the medium. The reversible effects of slightly acid pH vary with the temperature in the manner of an inhibitor (Type I) that acts independently of the normal, reversible denaturation equilibrium (K ₁) of the enzyme. The per cent inhibition caused by a given acid pH in relation to the luminescence intensity at optimum pH, is much greater at low temperatures, and decreases as the temperature is raised towards the optimum temperature. The observed maximum intensity of luminescence is thus shifted to slightly higher temperatures by increase in (H⁺). The apparent activation energy of luminescence is increased by a decrease in pH. The value of ΔH‡ at pH 5.05 was calculated to be 40,900 calories, in comparison with 20,700 at a pH of 6.92. The difference of 20,200 is taken to represent an estimate of the heat of ionization of ALH in the activation process, and compares roughtly with the 14,000 calories estimated for the same process, by analyzing the data from the point of view of hydrogen ions as an inhibitor. The decreasing temperature coefficient for luminescence in proceeding from low temperatures towards the optimum is accounted for in part by the greater degree of ionization of ALH. At the optimum temperature and acid reactions, pressures up to about 500 atmospheres retard the velocity of the luminescent oxidation. At the same temperature, with decrease in hydrogen ion concentration, the pressure effect is much less, indicating a considerable volume increase in the process of ionization and activation. In the extremely alkaline range, beyond pH 9, luminescence is greatly reduced, as compared with the intensity at neutrality, and under these conditions pressure causes a pronounced increase in intensity, presumably by acting upon the reversible denaturation equilibrium of the protein enzyme, A. Sulfanilamide, in neutral solutions, acts on luminescence in a manner very much resembling that of hydrogen ions at acidities between pH 4.0 and pH 6.5. Like the hydrogen ion equilibrium, the sulfanilamide equilibrium involves a ratio of approximately one inhibitor molecule to one enzyme molecule. The heat of reaction amounts to about 11,600 calories or more in a reversible combination that evidently evolves heat. Like the action of H ions, sulfanilamide causes a slight shifting of maximum luminescence intensity in the direction of higher temperatures, and an increase in the energy of activation. The effect of sulfanilamide on the growth of broth cultures of eight species of luminous bacteria indicates that there is no regular relationship among the different organisms between the concentration of the drug that prevents growth, and that which prevents luminescence in the cells which develop in the presence of sulfanilamide. p-Aminobenzoic acid (PAB) antagonizes the sulfanilamide inhibition of growth in luminous bacteria, and the cultures that develop are luminous. When (PAB) is added to cells from fully developed cultures, it has no effect on luminescence, or causes a slight inhibition, depending on the concentration. With luminescence partly inhibited by sulfanilamide, the addition of PAB has no effect, or has an inhibitory effect which adds to that caused by sulfanilamide. Two different, though possibly related, enzyme systems thus appear to limit growth and luminescence, respectively. The possible mechanism through which both the inhibitions and the antagonism take place is discussed. The irreversible destruction of the luminescent system at temperatures above that of the maximum luminescence, in a medium of favorable pH to which no inhibitors have been added, proceeds logarithmically with time at both normal and increased hydrostatic pressures. Pressure retards the rate of the destruction, and the analysis of the data indicates that a volume increase of roughly 71 cc. per gm. molecule at 32° C. takes place in going from the normal to the activated state in this reaction. At normal pressure, the rate of destruction has a temperature coefficient of approximately 90,000 calories, or about 20,000 calories more than the heat of reaction in the reversible denaturation equilibrium. The data indicate that the equilibrium and the rate process are two distinct reactions. The equation for luminescence intensity, taking into account both the reversible and irreversible phases of the reaction is given below. In the equation b is a proportionality constant; k'' the rate constant of the luminescent reaction; A₀ the total luciferase; A_0i the total initial luciferase at time t equals 0; k_n the rate constant for the destruction of the native, active form of the enzyme; k_d the rate constant for the destruction of the reversibly denatured, inactive form; t the time; and the other symbols are as indicated above: See PDF for Equation For reasons cited in the text, k_n evidently equals k_d. Urethane and alcohol, respectively, act in a manner (Type II) that promotes the breaking of the type of bonds broken in both the reversible and irreversible reactions and so promotes the irreversible denaturation. This result is in contrast to the effects of sulfanilamide, which at appropriate concentrations may give rise to the same initial inhibition as that caused by urethane, but remains constant with time. The inhibition caused by urethane and alcohol, respectively, increases as the temperature is raised. As a result, the apparent optimum is shifted to lower temperatures, and the activation energy for the over-all process of luminescence diminishes. An analysis for the approximate heat of reaction in the equilibrium between these drugs and the enzyme, indicates 65,000 calories for urethane, and 37,000 for alcohol. A similar analysis with respect to the effect of hydroxyl ions as the inhibitor gives 60,300 calories. The effects of alcohol and urethane are sensitive to hydrostatic pressure. Moderate inhibitions at optimum temperature and pH, caused by relatively small concentrations of either drug, are completely abolished by pressures of 3,000 to 4,000 pounds per square inch. At optimum temperature and pH, increasing concentrations of alcohol caused the apparent optimum pressure for luminescence to shift markedly in the direction of higher pressures. Analysis of the data with respect to concentration of alcohol at different pressures indicated that the ratio of alcohol to enzyme molecules amounted to approximately 4, at 7,000 pounds, but only about 2.8 at normal pressures. This phenomenon was taken to indicate that more than one equilibrium is established between the alcohol and the protein. A similar interpretation was suggested in connection with the fact that analysis of the relation between concentration of urethane and amount of inhibition at different temperatures also indicated a ratio of urethane to enzyme molecules that increased with temperature in the equilibria involved. Analysis of the data with respect to pressure and the inhibition caused by a given concentration of alcohol at different temperatures indicated that the volume change involved in the combination of alcohol with the enzyme must be very small, while the actual effect of pressure is apparently mediated through the reversible denaturation of the protein enzyme, which is promoted by alcohol, urethane, and drugs of similar type.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

THE OXIDATION-REDUCTION POTENTIAL OF THE LUCIFERIN-OXYLUCIFERIN SYSTEM

The oxidation-reduction potential of the Cypridina luciferin-oxyluciferin system determined by a method of "bracketing" lies somewhere between that of anthraquinone 2-6-di Na sulfonate (E_o ^'' at pH of 7.7 = –.22) which reduces luciferin, and quinhydrone (E_o ^'' at pH of 7.7 = +.24), which oxidizes luciferin. Systems having an E_o ^'' value between –.22 and +.24 volt neither reduce oxyluciferin nor oxidize luciferin. If the luciferin-oxyluciferin system were truly reversible considerable reduction and oxidation should occur between –.22 and +.24. The system appears to be an irreversible one, with both "apparent oxidation" and "apparent reduction potentials" in Conant''s sense. Hydrosulfites, sulfides, CrCl₂, TiCl₃, and nascent hydrogen reduce oxyluciferin readily in absence of oxygen but without luminescence. Luminescence only appears in water solution if luciferin is oxidized by dissolved oxygen in presence of luciferase. Rapid oxidation of luciferin by oxygen without luciferase or oxidation by K₃Fe(CN)₆ in presence of luciferase but without oxygen never gives luminescence.     

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

The incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T_m) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K_m and k_cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T _m) and the urea midtransition concentration (C _m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T _m and C _m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.     

A slow fluctuation in the molecular conformation of a soya bean trypsin inhibitor

The kinetics of the hydrogen-deuterium exchange reaction in a trypsin inhibitor (Kunitz) from soya bean have been followed by infrared absorption measurements in aqueous solutions at various temperatures and pH values. It was found that, in every case, 49% of the total peptide hydrogen atoms exchange relatively slowly. This amount corresponds to 83 peptide groups per molecule, and this is considered to be equal to the number of peptide NH groups involved in hydrogen bonds with the carbonyls of other peptide groups in the protein molecule in its native form. Each rate constant (k) determined at pH 2.75 for this category of the NH groups is in good agreement with the value expected from an idea that the breaking of the peptide-peptide hydrogen bonds takes place very slowly, and that this is the rate-determining process in the hydrogen-deuterium exchange reaction. Thus, by ultraviolet absorption measurements at 297 nm, the equilibrium constant of the native and denatured forms has been determined in the temperature range from 42 to 53.5 °C, as well as the reaction rate of reaching equilibrium from an off-equilibrium state. From these data the rate constant (k₁) of the denaturation reaction is determined, and the k₁ value is found to be practically equal to the hydrogen exchange rate constant (k). The Arrhenius plot of this rate constant (k) gives a straight line in the 25 to 55 °C region, and this gives a value of 48.6 kcal/mol for the activation energy of the denaturation reaction. The rate of this reaction is found to be very low at 25 °C; its half-life is about eleven days. Infrared absorption spectra observed in the amide I region suggest that the very slow denaturation of this protein is accompanied by a conformation change from an α-helix to a β-form. The number of the peptide groups involved in this α → β change is estimated to be 9 ± 3.     

In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH₂)-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes—methylviologen-nitrate reductase and NADH-ferricyanide reductase—also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH₂-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH₂-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t_½ [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH₂ do not donate electrons to the same site of the nitrate reductase protein.     

Chemical enthalpy-entropy compensation effect in the hydrolysis of p-nitrophenyl carboxylates catalyzed by alkaline mesentericopeptidase

The temperature dependence of the hydrolysis of p-nitrophenyl carboxylates with general formula H(CH₂)_nCOOC₆H₄NO₂ catalyzed by alkaline mesentericopeptidase has been studied (n varying from 1 to 7, temperature range 2–30°C, pH 8.80, 5 vol% dimethylsulfoxide). The activation parameters of the deacylation step depend on the length of the hydrophobic side chain of the substrate molecule ( , , and decrease by 2.0 kcal/mol, 4.9 kcal/mol, and 10 eu, respectively, as the length of the acyl carbon chain increases from n = 1 to n = 4). The following criteria were applied to establish a chemical enthalpy-entropy compensation effect: (a) Exner's plot of log vs : (b) Petersen's plot of log, k/T vs 1/T; (c) Exner's statistical treatment in coordinates log k vs 1/T; (d) according to Krug et al. (ΔH^≠ vs ΔG_Thm^≠). By use of all the above-mentioned criteria the existence of a chemical enthalpy-entropy compensation effect was proved with an isokinetic temperature β of about 470°K, which is significantly higher than the average experimental temperature.     

Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Acid Tolerance Response Induced by Organic Acids at 20°C: Optimization and Modeling

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20°C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (t_s), the log-linear decline (k_max), and the magnitude of a critical component (C). The magnitude of k_max gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.     

Thermotropic gelation of ovalbumin: 2. Boundary conditions on the formation and the concentration dependence of equilibrium elastic modulus for thermotropic ovalbumin gels

Studies were made on the boundary conditions for thermotropic ovalbumin gelation at pH within the range 2.5 to 10.0. The pH dependence of the gelation threshold, C₀, and denaturation temperature, T_d, were obtained. The dependence C₀(pH) has a sharp minimum close to the isoelectric point (pl). Over pH range 2.5 to 4.0 the dependence T_d(pH) is linear; although above pI it shows unusual behaviour. T_d increases smoothly, becoming a constant value (T_d=80°C) at pH 7. Analysis of the temperature dependence of Leu's line integral intensity in the p.m.r. spectrum of ovalbumin shows that the temperature threshold of thermotropic gelation closely approximates to T_d. A diagram for the state of an ovalbumin -water system was constructed in temperature-concentration-pH coordinates. The dependences of the initial shear modulus for thermotropic ovalbumin gels on the concentration (0.06≤C≤0.25g/cm³ were obtained at pH 4.0, 7.0, 8.5, 10.0. They are equivalent to the concentration dependence of the equilibrium elastic modulus E_e(C). The dependences obtained may be reduced to the theoretical master dependence of Hermans, $\text{E}{}_{\mathrm{e}}\text{(rm}\text{C}\text{?}\text{)}$, where $\text{C}\text{?}\text{=}\text{C}\text{/}\text{C}{}_0$ is the reduced concentration. Hermans' theory, based o the model for random cross-linking of linear identical macromolecules without cyclization, adequately describes the equilibrium elastic properties of thermotropic ovalbumin gels.     

Purification and Characterization of an Extracellular,Thermo-Alkali-Stable,Metal Tolerant Laccase from Bacillus tequilensis SN4

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k _cat/K _m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu²⁺, Co²⁺, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.     

GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509

A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K _m and k _cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s^?1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K _m and k _cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s^?1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K _m and k _cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s^?1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.     

THERMAL DEPOLARIZATION OF FLUORESCENCE FROM POLYTENE CHROMOSOMES STAINED WITH ACRIDINE ORANGE

The degree of polarization of fluorescence from stretched Chironomus thummi polytene chromosomes, stained with low concentrations of acridine orange (AO), decreases with increasing temperature. The "half temperature" of this decrease (T_½R) is lower than the expected DNA thermal denaturation temperature (T_m) by about 20°C. T_½R is lowered as histone is removed from chromosomes. Balbiani ring regions of the fourth chromosome have T_½R's much lower than other regions, and nearly as low as chromosomes which had been extensively pretreated with trypsin to remove histone and other proteins. Measurements of the thermal change in the rotational diffusion rate of AO in solution with DNA indicate that the temperature at which the DNA-AO bonding changes from a "rigid" to a "loose" mode varies with the GC percentage of the DNA, and in the same fashion as T_m, although 20°C lower.     

A Model for the Action of Vinblastine in Vivo

A model for the action of vinblastine (VLB) on cells multiplying exponentially in vivo with a generation time, T_G, has been derived. It is based on the assumption that cells attempting to pass through mitosis in the presence of VLB lose their proliferative capacity and that this lethal effect occurs only when the cells are exposed to a concentration of VLB which is above a critical value, C_k. The model leads to two predictions. First, that the percentage of cells surviving at any time after exposure to a dose, D, of VLB is 100% if D < D_k and decreases to 0% after a time, T_G, following a dose D ≥ D_k·2^{T G/T1/2}, where D_k represents the dose of VLB required to produce the concentration C_k, and T_1/2 is the half-life of the VLB in vivo. Second, that the time, T_G, at which the percentage of cells surviving an exposure to VLB, at doses greater than D_k·2^{U G/T1/2}, decreases to zero should be equal to the generation time of the cells. Both of these predictions were confirmed experimentally which indicates that the model adequately explains the action of VLB in vivo.     

Monte Carlo simulation of denaturation of adsorbed proteins

Denaturation of model proteinlike molecules at the liquid–solid interface is simulated over a wide temperature range by employing the lattice Monte Carlo technique. Initially, the molecule containing 27 monomers of two types (A and B) is assumed to be adsorbed in the native folded state (a 3 × 3 × 3 cube) so that one of its sides is in contact with the surface. The details of the denaturation kinetics are found to be slightly dependent on the choice of the side, but the main qualitative conclusions hold for all the sides. In particular, the kinetics obey approximately the conventional first-order law at T > T_c (T_c is the collapse temperature for solution). With decreasing temperature, below T_c but above T_f (T_f is the folding temperature for solution), deviations appear from the first-order kinetics. For the most interesting temperatures, that is, below T_f, the denaturation kinetics are shown to be qualitatively different from the conventional ones. In particular, the denaturation process occurs via several intermediate steps due to trapping in metastable states. Mathematically, this means that (i) the transition to the denatured state of a given molecule is nonexponential, and (ii) the denaturation process cannot be described by a single rate constant k_r. One should rather introduce a distribution of values of this rate constant (different values of k_r correspond to the transitions to the altered state via different metastable states). Proteins 30:168–176, 1998. © 1998 Wiley-Liss, Inc.     

Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353

One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V_max) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V_max during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V_max during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1°C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at ~35°C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.