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At least 1,153 records · Page 64Linked to original sources
PubMed1975

Hydrogen ion titration of lysozyme in alcohol-water solutions.

H+ titration curves of hen egg-white lysozyme were obtained at 0.15 I in the presence of small amounts (less than 15%) of methanol, ethanol and n-propanol. The acidity constants of two groups (whose pK values in water are, respectively, 42 and 3.5) are increased in water-alcohol mixtures in comparison to water. From the evaluation of these constants as a function of alcohol concentration and hydrocarbon chain length, it is suggested that these alcohols interact specifically with lysozyme. As pK values of 4.2 and 3.5 in water are generally assigned to Asp-101 and Asp-52 respectively, it seems that interaction occurs within the active site of the enzyme.

1-Propanol↗
PubMed1978

The enthalpy titration of troponin C with calcium.

Microcalorimetric titrations have been used to study the binding of Ca2+ to troponin C, the Ca-binding component of troponin. Troponin C was extracted from rabbit skeletal muscle and Ca2+ was added to Ca-free troponin C in the presence of 1 mM Mg2+ at pH 8.83 at 10 degrees C. In these conditions proton exchanges of troponin C on Ca-binding are negligible. In case of troponin C there is neither aggregation nor dissociation when Ca2+ is added, a phenomenon controlled by bound Ca, which caused some difficulty in the analysis of results for troponin. Using an iterative curve fitting procedure the following parameters of two classes of Ca-binding sites have been determined from the calorimetric results: number of binding sites in 1st class, 1.81; in 2nd class, 0.69; log of binding constant in 1st class, 7; in 2nd class, 5.0; standard enthalpy change for 1st class, -10.00 kJ . mol-1; that in 2nd class, -25.64. The standard free energy and entropy changes have been calculated from these values. The results indicate that the standard entropy change for Ca2+-troponin C interaction is positive and strikingly resembles the values for the interaction of organic sequestering agents such as glycol-EDTA with Ca2+.

Animals↗
PubMed1980

Affinity titration curves: determination of dissociation constants of lectin-sugar complexes and of their pH-dependence by isoelectric focusing electrophoresis.

By performing electrophoresis perpendicular to a stationary pH gradient (pH-mobility curves) in polyacrylamide gels containing a specific ligand either covalently fixed or entrapped in the gel matrix, it is possible to measure dissociation constants (Kd) and their pH-dependence in the pH range 3.5-10. The present technique, called 'affinity titration curves', is an extension of 'affinity electrophoresis'. This system has been applied to the study of the interaction between lectins and sugars: lectin from Ricinus communis seeds and alpha-D-galactose, and lectin from Lens culinaris seeds and alpha-D-mannose. The pH-dependence of Kd values indicated a more rapid decrement of affinity of both lectins for their ligands at acidic pH as compared to alkaline pH. For both lectins, maximum affinity was found in the pH range 7-8. Since the ionic strength of focused carrier ampholytes is 100-200-times lower than in conventional electrophoresis, the Kd values found by the present method are generally lower than the same values obtained by affinity electrophoresis.

Carbohydrates↗
PubMed1989

Titration of variant DNA sequences differing by a single point-mutation by selective dot-blot hybridization with synthetic oligonucleotides.

A dot-blot hybridization procedure with synthetic oligonucleotide probes is reported, which allows the quantitative titration in genomic DNA of variant forms of repeated genes differing by a single nucleotide change. It involves the utilization of a pair of 22-base long oligonucleotides matching the two variant sequences and the choice of an hybridization temperature very close to the Td of the oligonucleotide/DNA duplexes. The selectivity is achieved through a competition between the cognate labeled and the non-cognate unlabeled probes in the hybridization mixture.

Base Sequence↗
PubMed1977

A dose ratio comparison of the interaction between morphine and cyclazocine with naloxone in rhesus monkeys on the shock titration task.

Dose-response curves for morphine (0.5-20 mg/kg) were obtained in 4 rhesus monkeys performing the discrete trials shock titration task. Naloxone (5-500 microgram/kg) uniformly produced a dose dependent, parallel shift of the morphine dose-response curves to right. Plots for the 4 animals of the Log (dose ratio--1) versus --Log (naloxone) revealed that the line had a slope of -0.96. The value of the pA2 was 7.2. Similar experiments were carried out in two rhesus monkeys using cyclazocine. Naloxone (50-1000 microgram) similarly produced a rightward dose dependent, parallel shift of the cyclazocine dose response curves. Unlike the results obtained with morphine, the pA2 for naloxone obtained with cyclazocine was 6.3 and the slope of the dose ratio line was -1.6.

Animals↗
PubMed1983

Effects of ketocyclazocine and ethylketocyclazocine on electric shock titration.

The effects of two putative kappa agonists, ketocyclazocine and ethylketocyclazocine, on electric shock titration in squirrel monkeys were determined. Drugs were tested alone, in combination with naloxone and following the development of morphine tolerance. Both ketocyclazocine and ethylketocyclazocine produced biphasic dose-effect curves. Low doses decreased whereas high doses increased the intensity at which monkeys maintained the shock. Naloxone antagonized the effects of ketocyclazocine and ethylketocyclazocine; however, the dose of naloxone required to antagonize their effects was much larger than the dose required to antagonize equivalent effects of morphine. When the effects of ethylketocyclazocine were redetermined in monkeys tolerant to morphine, the ethylketocyclazocine dose-effect curve was shifted to the left whereas the morphine curve was shifted to the right. It was concluded that the effects of the kappa agonists can be differentiated from morphine by the shape of their dose-effect curve, the ease with which they are antagonized by naloxone and the lack of cross tolerance to morphine.

Animals↗
PubMed1984

Quantitative analysis of the interaction between the agonist and antagonist isomers of picenadol (LY150720) on electric shock titration in the squirrel monkey.

The opioid mixed agonist-antagonist picenadol (LY150720) is a racemate whose resolution results in a highly stereospecific separation of opioid agonist and antagonist activity. Attenuation of the antinociceptive effects of the agonist (dextro) isomer LY136596 by the antagonist (levo) isomer LY136595 was evaluated quantitatively in squirrel monkeys responding under a schedule of electric shock titration through the use of a dose-ratio analysis. LY136596 (0.3-3.0 mg/kg) produced a dose-related increase in the intensity at which monkeys maintained the shock. Increases in shock intensity produced by LY136596 were antagonized by LY136595 (0.1-10.0 mg/kg); dose-response curves for LY136596 were shifted to the right in a parallel manner by increasing doses of LY136595. An apparent pA2 (Schild) plot obtained from these data yielded a line with a slope of -0.60 +/- 0.05 and an apparent pA2 value of 5.67 +/- 0.07. These data support previous suggestions that the antinociceptive activity of picenadol (LY150720) resides in the d-isomer (LY136596) and that the l-isomer (LY136595) acts to limit the analgesic efficacy of the racemate.

Analgesics↗
PubMed1984

EPR titration of ovine prostaglandin H synthase with hemin.

To characterize further the prosthetic group of PGH synthase (EC 1.14.99.1), titrations of the apoenzyme with hemin were investigated by EPR. The first hemin bound per polypeptide showed an EPR signal at g = 6.7 and 5.3 (rhombicity 9%) and was tentatively assigned to the hemin effective as prosthetic group of PGH synthase. Additional hemin bound showed a less rhombic signal (g = 6.3 and 5.8, rhombicity 3%) presumably due to nonspecific hydrophobic binding sites not effective in catalysis.

Animals↗
PubMed1985

Further comments on the logic of the application of uncoupler- inhibitor titrations for the elucidation of the mechanisms of energy coupling.

Following the appearance of two papers in this journal commenting on the logic of the application of uncoupler-inhibitor titrations as a means of discriminating between 'delocalized' and 'localized' chemiosmotic mechanisms [(1984) FEBS Lett. 176,79-82; (1985) FEBS Lett. 186, 8-10], and in contrast with the arguments presented there and elsewhere, we show that in a linear model the increase in delta mu H which accompanies partial inhibition of the ATPases always leads to a relatively higher decrease of the rate of ATP synthesis by a given concentration of uncoupler in the presence of an ATPase inhibitor than in its absence. This is due to the fact that the same titre of uncoupler leads to a higher dissipative H+ flow in the presence of inhibitor, since the driving force delta mu H is higher.

Adenosine Triphosphatases↗
PubMed1989

Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction.

The cytochrome-bo quinol oxidase of Escherichia coli contains a high-spin b-type heme (cytochrome o), a low-spin b-type heme (cytochrome b) and copper. The EPR signal from cytochrome o is axial high spin and when titrated potentiometrically gives a bell-shaped curve. The low-potential side of this curve (Em7 approx. 160 mV) corresponds to the reduction/oxidation of the cytochrome. The high-potential side (Em7 approx. 350 mV) is proposed to be due to reduction/oxidation of a copper center; in the CuII form tight cytochrome o-copper spin coupling results in a net even spin system and loss of the EPR spectrum. Optical spectra of the alpha-bands of the reduced cytochromes at 77 K show that cytochrome b has its maxima at 564 nm when cytochrome o is oxidized but that this shifts to 561 nm when cytochrome o (max. 555 nm) is reduced. Both a heme-copper (cytochrome o-CuII) and a heme-heme (cytochrome o-cytochrome b) interaction are indicated in this quinol oxidase. These results indicate that cytochrome-bo quinol oxidase has a binuclear heme-copper catalytic site and suggest striking structural similarity to subunit I of the cytochrome aa3 system.

Copper↗
PubMed1991

Electrochemical redox titration of cofactors in the reaction center from Rhodobacter sphaeroides.

The electrochemical redox poising of the primary electron donor P and of the quinone electron acceptor(s) Q in isolated reaction centers from Rhodobacter sphaeroides in an ultra-thin-layer electrochemical cell, monitored by chronoamperometry and by spectroscopy in the visible/near-infrared region, is reported. Electrical application of a redox potential of +0.4 V (vs. Ag/AgCl/3 M KCl) leads to quantitative formation of the pi-cation radical of P within a few minutes. The oxidized product can be re-reduced to the neutral species by application of 0 V, and full reversibility is maintained over many cycles. By poising at a series of intermediate potentials, a titration curve for the 865 nm P band was obtained, which could be fitted to a Nernst function with Em = 0.485 vs. SHE and n = 0.96. By application of negative potentials (-0.2 V and -0.45 V vs. Ag/AgCl/3 M KCl), the quinone electron acceptors were reversibly reduced as demonstrated by the shift of bacteriopheophytin absorption and drastically changed kinetics of charge recombination. The use of this thin-layer electrochemical technique for the determination of midpoint potentials, for the investigation of redox-poised electron transfer reactions as well as for spectroscopy in the mid-infrared region is discussed.

Electrochemistry↗
PubMed1995

NMR-titrations with complexes between ds-DNA and indole derivatives including tryptophane containing peptides.

It is shown that NMR titrations can be used on a quantitative basis to derive binding constants and binding modes of ds-DNA ligand complexes from several signals. The results are partially at variance with literature conclusions; they can be interpreted by additive and independent contributions of electrostatic interactions between DNA phosphate and ammonium centers in the side chain of the ligands, and of very weak stacking contributions of the indole rings. While gramine and tryptamine are found to intercalate, tryptophane-containing peptides do so only, if the tryptophane is flanked by at least two lysine units.

Alkaloids↗
PubMed1978

An improved method for detection and titration of antibodies cytophilic for macrophages.

A new microtechnique for detection and titration of antibodies cytophilic for homologous macrophages is introduced and experiments designed to determine the optimum conditions for its performance reported. The technique employs a series of small chambers formed on a microscope slide by the application of a plastic film in which holes have been punched. The procedure uses 5 x 10(4) cells and 25 microliter of serum per chamber, and each test can be completed in 2 h. A permanent preparation results which can be examined as and when convenient.

Animals↗
PubMed1980

A large-scale radiometric micro-quantitative complement fixation test for serum antibody titration.

A micro-quantitative complement fixation (CF) procedure based on 51Cr release is described. The method employs 50% hemolysis as end point and the alternation equation to calculate the amount of complement involved in the hemolytic reaction. Compared to the conventional CF tests, the radiometric procedure described here is very precise and consistently reproducible. Also, since only 3 4-fold dilutions of sera are used for the titration of antibodies over a wide range of concentrations, the test is very concise and is economical to perform. Its format is amenable to automation and computerization. This radiometric CF procedure is thus most useful for large-scale immunological research and epidemiological surveillance studies.

Animals↗
PubMed1980

Lectin immuno tests: quantitation and titration of antigens and antibodies using lectin-antibody conjugates.

We have investigated the possibility of using lectin-antibody conjugates as general reagents in immunological procedures requiring a labeled antigen or antibody. Using these conjugates, labeling is achieved through saccharide binding sites of lectins which operate as acceptors for glycoconjugate marker substances added secondarily. Marker substances used in this work were enzymes, radioactively labeled glycoconjugates and erythrocytes, but other markers can also be used. Using the first two markers, antigens and antibodies were determined with accuracy and sensitivity equal to those of conventional enzyme or radioimmunoassays. Using erythrocytes as a marker, a simple erythro-adsorption procedure, possibly followed by hemolysis, has been developed which allowed the titration of antigens and antibodies to be carried out with a sensitivity at least equal to enzyme or radioimmunoassays.

Animals↗
PubMed1983

A rapid and reliable in vivo method for anti-GPLA (class I and class II antigens) antibody titration and GPLA typing.

A reverse Arthus reaction (RAR) may be successfully used in guinea pigs to detect histocompatibility alloantigens of the GPLA type. Epidermal cells carry class I GPLA antigens, and Langerhans cells also bear class II alloantigens. It is therefore possible to elicit an RAR by intradermal injection of relevant alloimmune sera in the skin of guinea pigs of known GPLA haplotype. RAR is detected by increased vascular permeability due to IgG1 antibody and hemorrhage due to IgG2 antibody. Compared with an in vitro protein A-rosetting method RAR is easier and quicker. It proved more sensitive for class II antigens in which Langerhans cells are the target for anti-class II antibody and rather less sensitive for class I antigens. RAR is a convenient method for following the course of GPLA alloimmunization, allowing titration of antibodies against both classes of antigen. It may also be used to type guinea pigs of unknown GPLA haplotype.

Animals↗
PubMed1983

Spot immunoprecipitate assay in gel compared with immune reactions in solution and applied to antibody titration.

Precipitation profiles of spot immunoprecipitate assay (SIA) reactions in gel are shown to have characteristics analogous to classic precipitin curves representing such immune reactions in solution. Thus SIA profiles of precipitated human albumin (HSA) and anti-HSA as checkerboard analysis permit: (1) determination of optimal proportions of antibody and antigen, (2) titration of antiserum as to antigen-binding capacity and (3) estimation of the concentration of specific antibody in an antiserum. The validity of the method was confirmed by similar results already reported for precipitin analysis of anti-HSA in solution. The stained protein assay method for determination of total protein used for the calculations had a reliable (r = 0.9979) working range for quantification between 0.1 and 0.83 microgram protein in 3 microliters samples (greater than or equal to 0.03 g/l). Study of SIA profiles in the extreme antibody excess region confirmed the validity of SIA quantification. The high sensitivity of SIA as compared with radial immunodiffusion is related to high ratios (8-9) of antibody to antigen; the reliable detection limit for SIA is 10 mg/l in a 3 microliters sample. SIA is easy to perform with simple laboratory equipment, allows measurement of any precipitable antigen with very small amounts of reactants, and gives results within an hour.

Albumins↗