New technique for measuring dynamic axonal transport and its application to temperature effects

A new technique was devised for the dynamic detection of the axoplasmic transport of β-radioactively labeled materials in which a semiconductor radiation detector was used as the β-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the β-radioactive distribution of axoplasmic transport could be measured in an axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 °C. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 °C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q₁₀ is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. This technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

Mapping heat exchange in an allosteric protein

Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein.     

40 Probing single molecule RNA folding using temperature and force

Nucleic acids can be unfolded either by temperature, such as in UV melting, or by mechanical force using optical tweezers. In UV melting experiments, the folding free energy of nucleic acids at mesophilic temperatures are extrapolated from unfolding occurring at elevated temperatures. Additionally, single molecule unfolding experiments are typically performed only at room temperature, preventing calculation of changes in enthalpy and entropy. Here, we present temperature-controlled optical tweezers suitable for studying folding of single RNA molecules at physiological temperatures. Constant temperatures between 22 and 37?°C are maintained with an accuracy of 0.1?°C, whereas the optical tweezers display a spatial resolution of ～1?nm over the temperature range. Using this instrument, we measured the folding thermodynamics and kinetics of a 20-base-pair RNA hairpin by force-ramp and constant force experiments. Between 22 and 37?°C, the hairpin unfolds and refolds in a single step. Increasing temperature decreases the stability of the hairpin and thus decreases the force required to unfold it. The equilibrium force, at which unfolding and refolding rates are equal, drops ～1?pN as temperature increases every 5?°C. At each temperature, the folding energy can be quantified by reversible work done to unfold the RNA and from the equilibrium constant at constant forces. Over the experimental temperature range, the folding free energy of the hairpin depends linearly on temperature, indicating that ΔH is constant. The measured folding thermodynamics are further compared with the nearest neighbor calculations using Turner’s parameters of nucleic acid folding energetics.     

Temperature-induced switching of the bacterial flagellar motor.

Chemotaxis signaling proteins normally control the direction of rotation of the flagellar motor of Escherichia coli. In their absence, a wild-type motor spins exclusively counterclockwise. Although the signaling pathway is well defined, relatively little is known about switching, the mechanism that enables the motor to change direction. We found that switching occurs in the absence of signaling proteins when cells are cooled to temperatures below about 10 degrees C. The forward rate constant (for counterclockwise to clockwise, CCW to CW, switching) increases and the reverse rate constant (for CW to CCW switching) decreases as the temperature is lowered. At about -2 degrees C, most motors spin exclusively CW. At temperatures for which reversals are frequent enough to generate a sizable data set, both CCW and CW interval distributions appear to be exponential. From the rate constants we computed equilibrium constants and standard free energy changes, and from the temperature dependence of the standard free energy changes we determined standard enthalpy and entropy changes. Using transition-state theory, we also calculated the activation free energy, enthalpy, and entropy. We conclude that the CW state is preferred at very low temperatures and that it is relatively more highly bonded and restricted than the CCW state.     

Relationship between temperature and growth rate of bacterial cultures.

The Arrhenius Law, which was originally proposed to describe the temperature dependence of the specific reaction rate constant in chemical reactions, does not adequately describe the effect of temperature on bacterial growth. Microbiologists have attempted to apply a modified version of this law to bacterial growth by replacing the reaction rate constant by the growth rate constant, but the modified law relationship fits data poorly, as graphs of the logarithm of the growth rate constant against reciprocal absolute temperature result in curves rather than straight lines. Instead, a linear relationship between in square root of growth rate constant (r) and temperature (T), namely, square root = b (T - T0), where b is the regression coefficient and T0 is a hypothetical temperature which is an intrinsic property of the organism, is proposed and found to apply to the growth of a wide range of bacteria. The relationship is also applicable to nucleotide breakdown and to the growth of yeast and molds.     

Free energy distributions in proteins.

Protein molecules in solution have a broad distribution of enthalpy states. A good approximation to the distribution function for enthalpy states can be calculated, using the maximum-entropy method, from the moments of the distribution that, in turn, are obtained from the experimental temperature dependence of the heat capacity. In the present paper, we show that the enthalpy probability distribution can then be formulated in terms of a free energy function that gives the free energy of the protein corresponding to a particular value of the enthalpy. By the location of the minima in this function, the free energy distribution graphically indicates the most probable values of the enthalpy for the protein. We find that the behavior of the free energy functions for proteins falls somewhere between two different cases: a two-state like function with two minima, the relative levels of the two states changing with temperature; and, a single-minimum function where the position of the minimum shifts to higher enthalpy values as the temperature is increased. We show that the temperature dependence of the free energy function can be expressed in terms of a central free energy distribution for a given, fixed temperature (which is most conveniently chosen as the temperature of the maximum in the heat capacity). The nature of this central free energy function for a given protein thus yields all of the thermodynamic behavior of that protein over the temperature range of the denaturation process.     

Solubility of Saturated Fatty Acids in Water at Elevated Temperatures

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230°C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160°C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.     

Refinement of noncalorimetric determination of the change in heat capacity, DeltaC(p), of protein unfolding and validation across a wide temperature range

The change in heat capacity, DeltaC(p), on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs-Helmholtz relationship to determine DeltaC(p) has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which DeltaG is zero, T(m), and change in enthalpy at T(m) are determined by thermal denaturation and DeltaC(p) is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of DeltaC(p) consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, T(m), and the van't Hoff enthalpy, DeltaH(vH), from a thermal denaturation, gives a reasonable estimate of DeltaC(p), albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting DeltaC(p), T(m), and DeltaH(vH) gives values for the last two parameters that are in excellent agreement with experimental values.     

Thermodynamics of the interaction of daunomycin with DNA

The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [³H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 × 10⁶ M^?1 (4°C) and 3.9 × 10⁵ M^?1 (65°C). The free energy change was ?8.2 to ?8.8 $\text{kcal}\text{mol}$, the enthalpy change ?5.3 $\text{kcal}\text{mol}$ and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appeals to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.     

New technique for measuring dynamic axonal transport and its application to temperature effects.

A new technique was devised for the dynamic detection of the axoplasmic transport of beta-radioactively labeled materials in which a semiconductor radiation detector was used as the beta-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the beta-radioactive distribution of axoplasmic transport could be measured in a axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 degrees D. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 degrees C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q10 is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. this technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

Temperature-dependence of rapid axonal transport in sympathetic nerves of the rabbit

Stop-flow techniques were used to determine how temperature affected the axonal transport of dopamine-β-hydroxylase (DBH) activity in rabbit sciatic nerves in vitro. These nerves were cooled locally to 2°C for 1.5 hr, which caused a sharp peak of DBH activity to accumulate above the cooled region. Accumulated DBH was then allowed to resume migration at various temperatures. From direct measurements of the rate of migration, we found that the axonal transport velocity of DBH was a simple exponential function of temperature between 13°C and 42°C. Over this range of temperatures, the results were well described by the equation: V = 0.546(1.09)^T, where V is velocity in mm/hr, and T is temperature in degrees centigrade. The Q₁₀ between 13°C and 42°C was 2.33, and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 kcal. Transport virtually halted when temperature was raised to 47°C, although only about half of the DBH activity disappeared during incubation at this temperature. Another transition occurred at 13°C; below this temperature, velocity fell precipitously. This was not an artifact peculiar to the stop-flow system since the rate of accumulation of DBH activity proximal to a cold-block also decreased abruptly when the temperature above the block was reduced below 13°C.     

Thermodynamics of anesthetic/protein interactions. Temperature studies on firefly luciferase.

Firefly luciferase is a soluble enzyme which is unusually sensitive to general anesthetics. The inhibition of the highly purified enzyme by three inhalational and three alcohol general anesthetics has been studied as a function of temperature, in the range from 5 to 20 degrees C. Inhibition constants Ki were determined at different temperatures, and van't Hoff plots of ln (Ki) versus reciprocal absolute temperature were found to be linear for all agents. Analysis of these plots gave values for the standard Gibbs free energy, enthalpy and entropy changes for transferring each anesthetic from water to the anesthetic-binding pocket on the protein. The most striking finding was that the enthalpy changes were much more negative for anesthetics binding to the protein than for binding to lipids or simple solvents. Furthermore, amongst the set of anesthetics studied, it was found that increasing potency correlated with favorable enthalpy rather than entropy changes. We discuss our results with respect to the molecular mechanisms underlying general anesthesia.     

Thermal denaturation of DNA in concentrated salt-free solutions: comparison of microcalorimetric and spectrophotometric data

Scanning microcalorimetry and spectrophotometry were used to study the dependence of melting enthalpy (delta Hm) and temperature (Tm) on DNA concentration in salt free solutions and on NaCl concentration in solutions with constant DNA concentration. This data is used to calculate the Manning's charge density parameter which is found to be equal 1.8. The linear dependence of Tm on the logarithm of DNA concentration in salt free solution was obtained. An approximate evaluation of dissociation degree in native DNA at different concentrations was made by comparison of straight lines in the Tm = f(lg CNaCl) and Tm = f(lg Cp) coordinates.     

Kinetic and thermodynamic studies on nitrobenzylthioinosine binding to the nucleoside transporter of chinese hamster ovary cells

The binding of [G-³H]nitrobenzylthioinosine to intact Chinese hamster ovary cells has been studied kinetically and thermodynamically. The association of nitrobenzylthioinosine with cells is a second-order process which proceeds at 24°C with a rate constant of 2·10⁷ M^?1·s^?1. Dissociation of the complex was characterized as a simple first-order process with rate constant on the order of 7·10^?3 s^?1. The quotient of these is comparable to the dissociation constant as measured in equilibrium binding studies, 2.2·10^?10 M. The temperature dependence of the rate of association indicated an Arrhenius activation energy of 8.4 kcal·mol^?1, while that of the equilibrium constant for dissociation indicated a standard enthalpy change of 8.8 kcal·mol^?1. The large increase in affinity of nitrobenzylthioinosine as compared to natural nucleosides is attributable to an entropy-driven interaction with the binding site. Thymidine, dipyridamole and papaverine each decrease the apparent dissociation constant for the nitrobenzylthioinosine-cell complex; the latter, inhibitors of nucleoside transport, decrease the rate of dissociation of the complex.     

Evaluation of data transformations used with the square root and schoolfield models for predicting bacterial growth rate.

A comparison was made between mathematical variations of the square root and Schoolfield models for predicting growth rate as a function of temperature. The statistical consequences of square root and natural logarithm transformations of growth rate use in several variations of the Schoolfield and square root models were examined. Growth rate variances of Yersinia enterocolitica in brain heart infusion broth increased as a function of temperature. The ability of the two data transformations to correct for the heterogeneity of variance was evaluated. A natural logarithm transformation of growth rate was more effective than a square root transformation at correcting for the heterogeneity of variance. The square root model was more accurate than the Schoolfield model when both models used natural logarithm transformation.     

Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction

Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.     

Evaluation of data transformations used with the square root and schoolfield models for predicting bacterial growth rate.

A comparison was made between mathematical variations of the square root and Schoolfield models for predicting growth rate as a function of temperature. The statistical consequences of square root and natural logarithm transformations of growth rate use in several variations of the Schoolfield and square root models were examined. Growth rate variances of Yersinia enterocolitica in brain heart infusion broth increased as a function of temperature. The ability of the two data transformations to correct for the heterogeneity of variance was evaluated. A natural logarithm transformation of growth rate was more effective than a square root transformation at correcting for the heterogeneity of variance. The square root model was more accurate than the Schoolfield model when both models used natural logarithm transformation.     

THE TOXIC ACTION OF COPPER ON NITELLA

1. Using the loss of turgidity of the cells as a criterion it is found that the toxicity curve of copper chloride with Nitella is sigmoid. An empirical equation can be constructed which will approximately fit the curve. 2. When the concentration of the copper chloride is varied the toxic effect varies as a constant, fractional, power of the concentration. This relation holds when the concentration is plotted against either (1) the time necessary to reach a given point on the ordinate of the survivor curve, (2) the maximum speed of toxic action as shown by the tangent to the survivor curve or (3) the first derivative of the equation which fits the survivor curve. 3. When the temperature is varied and the logarithm of the reciprocal of the time necessary to reach a given point on the survivor curves is plotted against the reciprocal of the absolute temperature the resulting figure consists of several intersecting curves. A hypothetical system is described which will give straight lines under normal conditions and curves when acted upon by a toxic agent.     

Theory and modelling of ethanol evaporative losses during batch alcoholic fermentation

Evaporative loss of ethanol during batch alcoholic fermentation has been modelled, employing modern concepts of kinetics and stoichiometry and the best available phase equilibrium thermodynamic data. Theoretical results demonstrate that loss is proportional to the second power of the sugar concentration utilized and that the logarithm of loss is proportional to reciprocal absolute temperature. Good agreement is demonstrated among the theory, the numerical model, and the literature results. A master correlation for predicting ethanol loss is presented.     

Interpretation of DSC data on protein denaturation complicated by kinetic and irreversible effects

Thermal denaturation of Kunitz soybean trypsin inhibitor (KTI) and ribulose-1,5-biphosphate carboxylase (RBPC) from tobacco leafs was studied by the method of high-sensitivity differential scanning calorimetry (HS-DSC). The dependence of the denaturation temperature on the heating rate reveals in the case of both proteins a non-equilibrium character of the denaturation transition in applied conditions. Developed kinetic approach allows the determination of an equilibrium transition temperature as well as the rate constants of denaturation and renaturation from the complex data of HS-DSC. This method was applied to the analysis of the pH-induced change of the conformational stability of KTI within pH range from 2.0 to 11.0. It allowed the determination of the pH dependencies: of the excess free energy of denaturation, of the activation enthalpy and entropy of denaturation as well as of the denaturation rate constant. Conclusions have been made suggesting the contribution of side-chain hydrogen bonds in the stabilisation of the native and activated states of KTI.