A new method for the determination of ligand dissociation rate constant of carboxyhemoglobin.

Concanavalin A (Con A) treatment of plasma membrane-enriched fractions from lactating mammary gland causes an activation of Mg⁺⁺-ATPase and an inactivation of 5′-nucleotidase. Both effects can be prevented by the presence of α-methylmannoside, and both exhibit cooperativity with Hill coefficients near 2. The cooperativity may arise from Con A effects on subunit interactions of the enzymes or by clustering of the enzyme molecules in the membrane, possibly induced by Con A. Investigations of these systems should be useful for developing an understanding of modes of action of Con A in complex phenomena.     

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 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 simple and rapid method for the reversible removal of lipids from a membrane-bound enzyme.

A simple, rapid and reproducible method for the reversible removal of lipids from a membrane-bound enzyme is described. Essentially, a membrane preparation containing (Na+ + K+)-dependent adenosine triphosphatase was extracted with the non-ionic detergent Lubrol WX in the presence of glycerol, and partial separation of protein from lipid was achieved with the use of only two centrifugations. About 74% of the endogenous phospholipid and 79% of the cholesterol were removed, concomitant with a virtually complete loss of ouabain-sensitive adenosine triphosphatase activity, but with retention of 60-100% of the K+-dependent phosphatase activity. The addition of pure phosphatidylserine re-activated the enzyme to more than 80% of the initial activity, and up to 30% of the protein was recovered. Excess of phosphatidylserine could be washed off the enzyme to give a stable 'reconstituted' preparation. The effects of variation in the experimental conditions were examined, and the results are discussed with respect to the possibility of adapting the method to the study of other lipid-dependent enzymes bound to membranes.     

A simple method to obtain a covalent immobilized phospholipase A(2).

In the present work, we obtained an immobilized phospholipase A(2) system through covalent coupling by using an acrylic polymer Eupergit C as support. The immobilized enzyme from cobra venom (Naja naja naja) showed good retention activity and excellent stability. Both properties are of great importance for biomedical applications such as hypercholesterolemia treatments.     

A simple method for calculating the statistical power for detecting a QTL located in a marker interval

We developed a simple method for calculating the statistical power for detecting a QTL located in an interval flanked by two markers. The statistical method for QTL detection is assumed to be the Haley and Knott's simple regression method of interval mapping. This method allows us to answer one of the fundamental questions in designing a QTL mapping experiment: What is the minimum marker density required to detect a QTL explaining a certain heritable proportion of the phenotypic variance (denoted by h(2)) with a power gamma under a Type I error alpha in an F(2) or other mating designs with a sample size n? Computing the statistical power only requires the ability to evaluate a non-central F-distribution function and the inverse function of this distribution.     

A simple method for the recovery of active oligomeric enzyme from its diluted solutions

A method for the recovery of active enzyme from its diluted solutions is described. It includes batch sorption on DEAE-Sephadex A-50 which follows elution or chromatography of an enzyme on a DEAE-Sephadex A-50 column. All purified aminoacyl-tRNA synthetases retain high activity without degradation of the protein. The method is very fast and convenient.     

A simple method for calculating the productivity of polyphenol separations by polymer-based chromatography

A simple method for calculating the productivity of chromatography processes was proposed based on the iso-resolution curve concept. The model separation system was polyphenol separations by polystyrene divinylbenzene resins with the ethanol–water mixture mobile phase. The distribution coefficient K was determined as a function of ethanol concentration I by linear gradient elution experiments. The HETP-mobile phase velocity u curves were determined as a function of I. Using K and HETP, the iso-resolution curve was calculated, from which the productivity was determined as a function of I. It was found that there is an optimum I, where the highest productivity with the minimum amount of mobile phase consumption is obtained.     

Photolysis method for determination of the tetramer-dimer dissociation constant of deoxyhemoglobin

A new method for determination of the tetramer-dimer dissociation constant Ku4.2 of deoxyhemoglobin is described. The method involves photolysis of hemoglobin solutions containing a few percent of bound CO (e.g. less than 3%). Under these conditions the nature of the observed CO rebinding is primarily determined by the properties of the dominant species, deoxyhemoglobin. The method makes use of the 30-fold difference in the rate constant describing CO binding to hemoglobin dimers and deoxyhemoglobin tetramers. Because of this large difference in rate constants CO rebinding is made significantly more rapid by the presence of even small concentrations of dimers. Treating this reaction as CO binding to a mixture of hemoglobin dimers and tetramers allows the determination of Ku4.2. Data is presented showing application of the method to human deoxyhemoglobin in the range from pH 9.5 to 11.2.     

A simple, rapid, and precise method for calculating the steroid hormone-receptor complex concentration in the presence of saturating levels of hormone by using "differential dissociation" techniques.

The steroid hormone-receptor complex concentrations measured by “differential dissociation” techniques have to be corrected to obtain the true concentrations of receptor binding sites (B_s). For the calculation of B_s, the parameters kn (product of the equilibrium association constant and the concentration of binding sites of the “nonspecific” component) and f (fraction of the nonspecific binding measured in the experimental estimates of bound ligand by a given technique), previously proposed by Blondeau and Robel (J. P. Blondeau and P. Robel, 1975, Eur. J. Biochem.55, 375–384) are important. A new parameter of interest, ? [$\text{?}\text{=}\text{knf}\text{(kn + 1)}$], is discussed. The measurement of this parameter ? for three “differential dissociation” techniques allows the comparison of their efficiency and their reliability under various conditions for hormone receptor measurement in cytosol. Charcoal and hydroxylapatite methods are more efficient than the Sephadex G-25 filtration method. It is demonstrated that the “isotopic dilution” correction generally used for the estimation of the background of a given technique may be incorrect whatever the method of correction. A new method, the “double concentration measurement,” is developed. This method is simple, rapid, and precise. It requires two receptor binding measurements at two different saturating concentrations of ligand. This method allows the measurement of the estradiol receptor binding activity from calf uterine cytosol, with an error of less than 5% in samples containing the receptor either free or previously complexed with radioactive hormone, even in the presence of very high concentrations (≤0.5 μm) of radioactive steroid.     

Acid dissociation constant, a potential physicochemical factor in the inhibition of the enzyme estrone sulfatase (ES)

We report the initial results of the synthesis and biochemical evaluation of a series of aminosulfonate based compounds of phenol and the determination of the pKa of the parent phenol in an attempt to investigate the role of this physicochemical factor in the irreversible inhibition of the enzyme estrone sulfatase (ES). The results of the study show that there is a strong correlation between the observed pKa and inhibitory activity. We postulate that the stability of the phenoxide ion, as indicated by the acid dissociation constant, is an important factor in the irreversible inhibition of this enzyme.