A method for determining the dielectric constant and the conductivity of membrane-bounded particles of biological relevance.

Numerical assessment is made regarding Pauly and Schwan's theory which describes the dielectric behavior of a suspension of "shell spheres" as a model of biological membrane-bounded particles. The results indicate that approximate expressions of the theory may give rise to serious errors when applied to particles smaller than about 1 mum in diameter. With a view to performing analysis according to a general expression of the theory, some of the characteristic responses of dielectric parameters upon changes in phase parameters are examined with particular reference to some numerical ranges of biological interest. On this basis a simplified and systematic procedure is proposed for estimating the phase parameters of particles whose shell phase can be regarded as non-conductive. As the application of the procedure proposed, a set of dielectric data of a synaptosome suspension is analyzed, so that the following three phase parameters are successfully determined: membrane capacitance (or shell phase dielectric constant), interval phase conductivity and internal phase dielectric constant. Some limitations of the procedure are discussed for the cases of conducting shells and small particles.     

A new method for determining the Michaelis constant

A new method is described for evaluating the parameters K(m) and V in the Michaelis-Menten equation, and is illustrated with experimental data from the literature.     

A method of determining the antioxidative activity of biological matter

A procedure is described for determining antioxidation activity of the water-soluble biological material. The procedure is based on the study of kinetics of the reduced 2.6-dichlorophenolindophenol oxidation by air oxygen both with and without the biological material as well as on calculation of the value for the constant of the biological material inhibition of 2.6-dichlorophenolindophenol oxidation as an index of the biological material antioxidation activity.     

A novel spectroscopic titration method for determining the dissociation constant and stoichiometry of protein-ligand complex.

We offer a new titration protocol for determining the dissociation constant and binding stoichiometry of protein-ligand complex, detectable by spectroscopic methods. This approach neither is limited to the range of protein or ligand concentrations employed during titration experiment nor relies on precise determinations of the titration "endpoint," i.e., the maximal signal changes upon saturation of protein by ligand (or vice versa). In this procedure, a fixed concentration of protein (or ligand) is titrated by increasing volumes of a stock ligand (or protein) solution, and the changes in the spectroscopic signal are recorded after each addition of the titrant. The signal for interaction between protein and ligand first increases, reaches a maximum value, and then starts decreasing due to dilution effect. The volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. This procedure has been tested for the interaction of avidin with a chromophoric biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid by following the absorption signal of their interaction at 500 nm. The widespread applicability of this procedure to protein-ligand complexes detected by other spectroscopic techniques and its advantages over conventional methods are discussed.     

A new graphic method for determining the rate constant for affinity of a ligand for its receptor]

A new method of determination of the equilibrium constant for a ligand binding to acceptor and evaluation of the number of binding sites on the acceptor molecules (or cells) is suggested. The method is simpler, more convenient, and more precise than Klotz's or Scatchard's method.     

A theoretical study of the dielectric constant of protein

The dielectric properties of a protein molecule were investigated by calculating a 'local dielectric constant' with the aid of normal mode analysis. This local dielectric constant was calculated from the electronic polarization of atoms and the orientational polarization of local dipoles. The former was obtained from atomic polarizations of the whole atoms in a protein molecule. The latter was determined from the fluctuation-dissipation theorem. The degree of dipole fluctuation was calculated from the positional fluctuation of each atom obtained by the normal mode analysis. Assuming a minimum volume for a continuum model, the resulting local dielectric constants ranged from 1 to 20 inside the protein.     

A method for determining tissue sulfate

A method for the determination of the inorganic sulfate present in rat liver homogenates has been developed. In order to determine sulfate, a protein-free extract is required. The classical protein precipitation methods of preparing protein-free extracts gave 2.5–40% recovery of added ³⁵SO₄^2?. Separation of the protein by ultrafiltration gave only 29% recovery when 0.15 m KCl was the homogenizing medium. A homogenization medium containing 0.154 m NH₄OH and 20 g EDTA per liter gave 102 ± 11% recovery of added ³⁵SO₄^2? when the protein was separated by ultrafiltration.     

Effect of some monohydric alcohols on the oxygen affinity of hemoglobin: relevance of solvent dielectric constant and hydrophobicity.

We studied the effect of methanol, ethanol, iso-propanol, and n-propanol on the reaction of hemoglobin with oxygen. The oxygen affinity was found to decrease with increasing alcohol concentration and alkyl group size; no detectable effect on Hill's constant was found. Difference spectroscopy indicated K_R not to be affected by the presence of alcohols; the lowered affinity was then attributed to an altered equilibrium between T and R conformations of hemoglobin. The results have been analyzed in such a way as to allow separation of electrostatic contributions to free energy difference between the T and R states from nonelectrostatic ones. The nonelectrostatic term has been attributed to protein–solvent hydrophobic interactions. Values of hydrophobic free energy are in good agreement with analogous data estimated by correlating different results previously reported in the literature.     

A method for determining a rate constant for biexponential processes using data on dynamics of accumulating the end product of a reaction

A new method the rate constant determination of some biexponential processes is suggested. The method is based on the asymptotic solution of transcendental irrational equations, described such processes. This method can be used when kinetics of the final product of reaction is known. The values of the rate constants obtained by suggested method are precise, if the data used for calculation are also precise. In the other case methods of mathematical statistics should be used for evaluation of the slopes of kinetics curves in the semi-logarithmic coordinates.