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PubMed1984

Design and testing of a new microcalorimetric vessel for use with living cellular systems and in titration experiments.

A new microcalorimetric vessel primarily intended for use with living cellular systems and in titration experiments has been designed and tested. The vessel, which forms a modular system, fits into an ampoule measuring cylinder of the LKB 'BioActivity Monitor'. It can be used with different sample cups, volume 1-3 ml, and can be equipped with different types of stirrer and sample holders for cellular materials. Experiments can be performed with or without medium perfusing through the vessel. Small quantities of reagents can be added to the sample compartment during the measurements. Stepwise calorimetric titrations can be performed by an automatic procedure. Test experiments reported include results of measurements with human T-lymphoma cells in stirred suspension and melanoma cells adhered to a polystyrene film in a stirred perfusing medium. Results from titration experiments where N-acetyl-D-alanine was bound to ristocetin A are reported, delta G degree' = -16.5 +/- 0.2 kJ mol-1 and delta H degree' = -32.1 +/- 0.4 kJ mol-1.

Calorimetry↗
PubMed1985

Use of the twin-cell differential titration calorimeter for binding studies. I. EDTA and its calcium complex.

The use of a twin-cell differential titration calorimeter is described which utilizes small volumes (1-3 ml) of modest concentrations of materials (0.001-0.01 M) and yields data of good precision. Operation is controlled by a microprocessor which regulates and controls the addition of reagents and collects and displays the data as time, temperature in volts, and the pH. Corrections for the titration of water are applied to the potentiometric data, and the thermal data are corrected for the initial temperature-time baseline, the changes in heat capacity, and the heat loss (or gain) to the external environment. Finally, the thermal signal is corrected for the heat derived from the formation of water due to the free hydrogen or hydroxyl ions present. The corrected data as pH, groups titrated and delta HT (kcal/mol) can then be used to obtain the parameters pK' and delta Hi involved with the equilibria by curve-fitting the observed data. The system has been applied to the ionization of EDTA and its calcium complex. The ionization constants, the heats of ionization, the stoichiometry of binding and the heat of binding have been determined and demonstrated to be in agreement with published values.

Calcium↗
PubMed1985

Cell titration assay for measuring blastogenesis of bovine lymphocytes.

The blastogenic response of bovine peripheral blood lymphocytes to phytohemagglutinin (PHA) and to microbial antigens was measured using a lymphocyte titration assay. Culture conditions, including lymphocyte concentrations, incubation periods and medium formulation, were established which produced linear or nearly linear responses over a range of cell concentrations. These conditions were established by testing lymphocytes from unimmunized cattle and from heifers infected with Brucella abortus with PHA and a B. abortus extract. Four cell concentrations in 2-fold increments were selected for measuring responses to PHA (3.125 X 10(3) to 2.5 X 10(4) cells/well) and to antigens (5.0 X 10(4) to 4.0 X 10(5) cells/well). The strength of response varied among animals and also over time for individual animals, but the titration assay allowed exponential proliferation to be distinguished from decline, which may have been due to overcrowding of microtiter wells, exhaustion of nutrients or induction of regulatory events. This assay provided a more reliable and discriminating method of evaluating lymphocyte proliferation responses than that achieved by single point assays. The displacement of the titration curves could be used to estimate the relative frequency of lymphocytes responding to antigens or mitogens.

Animals↗
PubMed1982

Procedure for titration of interleukin-2 based on direct observation of factor-dependent growth of cells.

A semi-quantitative procedure for titration of interleukin-2 (IL-2) without the use of radioactive labeling of IL-2 target cells was developed. The procedure is based on the observation that those cells cultivated in round-bottom wells formed visible cultures and that the dimension of these cultures decreased at higher dilutions of IL-2 in the medium. The end-point of the titration can be easily established by observing the cultures with a concave mirror placed under the titration plate. The procedure has been used for the determination of IL-2 during its purification and characterization.

Animals↗
PubMed1985

Virus enzyme-linked cell immunoassay (VELCIA): detection and titration of rotavirus antigen and demonstration of rotavirus neutralizing and total antibodies.

Virus enzyme-linked cell immunoassay (VELCIA) for detection and titration of rotavirus antigen has been developed. Wild-type porcine rotavirus antigen can be detected and titrated directly from the fecal material within 24 h. Porcine OSU strain can be titrated higher than 10(-8). The method has also been introduced for the demonstration of rotavirus neutralizing and total antibodies. In VELCIA the advantages of the cell culture system for virus isolation are combined with enzyme immunodetection and spectrophotometrical reading of the test.

Antibodies, Viral↗
PubMed1992

Triple inhibitor titrations support the functionality of the dimeric character of mitochondrial ubiquinol-cytochrome c oxidoreductase.

The ubiquinol-2 or duroquinol oxidoreductase activity of mitochondrial ubiquinol-cytochrome c oxidoreductase was titrated with combinations of antimycin, myxothiazol and N,N'-dicyclohexylcarbodiimide (DCCD). A statistical model has been developed that can predict the activity of the complex treated with all possible combinations of these inhibitors. On the basis of the measured titration curves the model had to accommodate interaction between the two promoters of the complex. The titrations confirm that treatment with DCCD results in the modification of a certain site in one of the two promoters of the bc1 dimer, thereby blocking one antimycin A binding site without inhibiting electron transfer. Modification of both antimycin A binding sites of the dimer is apparently required for inhibition of electron transfer through the complex, just as modification of both myxothiazol-binding sites is required for full inhibition. The conclusion can be drawn that mitochondrial ubiquinol-cytochrome c oxidoreductase is a functional dimer, consisting of electrically interacting protomers.

Animals↗
PubMed1996

Monte Carlo studies on potentiometric titration of (carboxymethyl)cellulose.

Monte Carlo simulations of the potentiometric titration are carried out for (carboxymethyl)cellulose in aqueous salt solutions by a previously developed method. A nearly elliptic cylinder with spherical ionizable groups is assumed as model of (carboxymethyl)cellulose molecule. The spherical charges with a hard core potential are adopted as mobile hydrated ions. A fairly satisfactory agreement of the titration curves with the experimental data is achieved by using reasonable molecular dimensions. Dependence of the calculated titration profiles on the molecular model and the characteristics of the system are discussed.

Journal Article↗
PubMed1992

pH titration curves of the desialylated human alpha 1-acid glycoprotein variants by combined isoelectrofocusing-electrophoresis: utilization in the development of a fractionation method for the protein variants by chromatography on immobilized metal affinity adsorbent.

Using a two-dimensional isoelectrofocusing (IEF)-electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then disialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5-9.5) and a narrow gradient (pH 5-8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5-5.5 and pH 7.5-9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.

Chromatography, Affinity↗
PubMed1993

Micellar acid-base potentiometric titrations of weak acidic and/or insoluble drugs.

The effect of various surfactants [the cationics cetyl trimethyl ammonium bromide (CTAB) and cetyl pyridinium chloride (CPC), the anionic sodium dodecyl sulphate (SDS), and the nonionic polysorbate 80 (Tween 80)] on the solubility and ionization constant of some sparingly soluble weak acids of pharmaceutical interest was studied. Benzoic acid (and its 3-methyl-, 3-nitro-, and 4-tert-butyl-derivatives), acetylsalicylic acid, naproxen and iopanoic acid were chosen as model examples. Precise and accurate acid-base titrations in micellar systems were made feasible using a microcomputer-controlled titrator. The response curve, response time and potential drift of the glass electrode in the micellar systems were examined. The cationics CTAB and CPC were found to increase considerably the ionization constant of the weak acids (delta pKa ranged from -0.21 to -3.57), while the anionic SDS showed negligible effect and the nonionic Tween 80 generally decreased the ionization constants. The solubility of the acids in aqueous micellar and acidified micellar solutions was studied spectrophotometrically and it was found increased in all cases. Acetylsalicylic acid, naproxen, benzoic acid and iopanoic acid could be easily determined in raw material and some of them in pharmaceutical preparations by direct titration in CTAB-micellar system instead of using the traditional non-aqueous or back titrimetry. Precisions of 0.3-4.3% RSD and good correlation with the official tedious methods were obtained. The interference study of some excipients showed that a preliminary test should be carried out before the assay of formulations.

Acids↗
PubMed1995

Propionyl-L-carnitine in intermittent claudication: double-blind, placebo-controlled, dose titration, multicenter study.

OBJECTIVES: The aim of this double-blind, placebo-controlled, dose titration, multicenter trial was to assess the efficacy and safety of propionyl-carnitine in intermittent claudication. BACKGROUND: Human and animal studies indicate that propionyl-L-carnitine increases carnitine content and improves energy metabolism in the ischemic skeletal muscle. METHODS: After a 2-week preliminary period to assess maximal walking distance, 245 patients were randomly assigned to receive propionyl-L-carnitine (n = 118) or placebo (n = 127). The initial oral dose of 500 mg twice daily was increased at 2-month intervals to 2 g/day and then to 3 g/day in patients showing improvement in treadmill performance < 30% over baseline. Efficacy analysis was conducted for the 214 patients who completed the 24 weeks of treatment by comparing the effect of placebo and propionyl-L-carnitine on day 180. RESULTS: Analysis of variance showed a significant improvement of 73 +/- 9% (mean +/- SE) in maximal walking distance with propionyl-L-carnitine (n = 99) compared with 46 +/- 6% for placebo (n = 115, p = 0.03). For distance walked at onset of claudication, propionyl-L-carnitine showed about double the improvement of placebo; however, the difference was not statistically significant. There were no changes in electrocardiographic and routine biochemical and hematologic tests that would indicate an adverse effect of propionyl-L-carnitine. Adverse events requiring drug discontinuation (11 in the propionyl-L-carnitine group, 3 in the placebo group) were unrelated to study medication. The dose titration design of the study also provided information on the dose-response relation. Slightly less than 67% of patients were expected to improve their maximal walking distance by at least 30%, assuming 2 g/day of propionyl-L-carnitine (95% confidence interval 0.51 to 0.70). The response rate during the entire titration course was significantly in favor of propionyl-L-carnitine compared with placebo. CONCLUSIONS: Although the precise mode of therapeutic action requires clarification, propionyl-L-carnitine, at a dose of 1 to 2 g/day, appears to be effective and well tolerated, with minimal adverse effects.

Administration, Oral↗
PubMed1993

Amino acid titration curves--misshapen or mislabeled?

Titration curves are frequently used to illustrate the change in charge of amino acids with pH, and as such are a fundamental part of most biochemistry courses. We have noticed that the graphs of titration curves appearing in many biochemistry textbooks are drawn incorrectly near the origin of the titration. This article demonstrates that this curve is misshapen, or, alternatively, that the axis of the graph is incorrectly labelled.

Amino Acids↗
PubMed2004

The development of a continuous isothermal titration calorimetric method for equilibrium studies.

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12-20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60-180 min to generate 20-30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba(2+) to 18-crown-6 and the binding of cytidine 2'-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.

Barium Compounds↗
PubMed2005

Ligand binding to one-dimensional lattice-like macromolecules: analysis of the McGhee-von Hippel theory implemented in isothermal titration calorimetry.

The theory developed by McGhee and von Hippel for ligand binding to a one-dimensional lattice-like macromolecule provides a closed analytical form in the Scatchard representation. The application of such theory has been complicated by two facts: (1) it has been practically reduced to binding techniques, such as equilibrium dialysis, in which the partition between bound and free concentrations of all reactant species are directly accessible and experimentally determined, but infrequently applied to other binding techniques, such as calorimetry or spectroscopy, in which the direct observable is a magnitude proportional to the advance of the binding reaction monitored along the titration experiment, and (2) Scatchard analysis, developed as a quantitative graphical method, is currently outdated and used only qualitatively because of its weaknesses, limitations, and deficiencies. However, a general exact method for applying such theory to titration techniques in a correct and precise manner, without any limitation, can be delineated. In this article, the theory of cooperative ligand binding to linear lattice-like macromolecules has been implemented in isothermal titration calorimetry for the first time. This technique provides a complete thermodynamic characterization of ligand binding, but it has been barely used properly for this type of system. The description, the analysis of the formalism, and practical guidelines are presented, with considerations for experimental design and data analysis.

Calorimetry↗
PubMed2004

Reductive titration of photosystem I and differential extinction coefficient of P700+ at 810-950 nm in leaves.

We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e-) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e- carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) [V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1-15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch (Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e- generated in PSII by the flash was measured as four times O2 evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700+ and PC+ components using the abovementioned oxidative titration method. The P700+ component was related to the absolute number of e- that reduced the P700+ to calculate the extinction coefficient. The effective differential extinction coefficient of P700+ at 810-950 nm was 0.40+/-0.06 (S.D.)% of transmittance change per micromol P700+ m(-2) or 17.6+/-2.4 mM(-1) cm(-1). The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (+/-14% S.D.) is mainly caused by light-scattering properties of the leaf.

Light↗
PubMed2004

Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range.

A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.

Escherichia coli Proteins↗
PubMed2006

Stabilization of the initial electrochemical potential for a metal-based potentiometric titration study of a biosorption process.

An interactive metal-based potentiometric titration method has been developed using an ion selective electrode for studying the sorption of metal cations. The accuracy of this technique was verified by analyzing the metal sorption mechanism for the biomass of Rhizopus arrhizus fungus and diatomite, two dissimilar materials (organic and mineral, strong sorbent and weak sorbent) of a different order of cation exchange capacity. The problem of the initial electrochemical potential was addressed identifying the usefulness of a Na-sulfonic resin as a strong chelating agent applied before the beginning of sorption titration experiments so that the titration curves and the sorption uptake could be quantitatively compared. The resin stabilized the initial electrochemical potential to -405+/-5 mV corresponding to 2 micro gl(-1) of lead concentration in solution. The amounts of lead sorbed by R. arrhizus biomass and diatomite were 0.9 mmol g(-1) (C(e)=5.16 x 10(-2)mM) and 0.052 mmol g(-1) (C(e)=5.97 x 10(-2) mM), respectively. Lead sorption by the fungal biomass was pinpointed to at least two types of chemical active sites. The first type was distinguished by high reactivity and a low number of sites whereas the other was characterized by their higher number and lower reactivity.

Adsorption↗
PubMed2006

Isothermal titration calorimetric study of calcium association to lipid bilayers: influence of the vesicle preparation and composition.

The association of Ca2+ ions with phospholipid bilayers was investigated using isothermal titration calorimetry. The study reveals that the binding enthalpy of these cations to bilayers formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) depends strongly on the method of preparation of the unilamellar vesicles. Extruded vesicles lead to an exothermic association, whereas sonicated ones lead to an endothermic association. In the later case, the calorimetric signal is sensitive to the length of the sonication period. It is proposed that a reorganization of the lipid bilayers under stress, obtained with sonicated small unilamellar vesicles, contributes to the calorimetric signal upon the titration with Ca2+. The analysis of the titrations indicates that, as expected, the nature of the association of Ca2+ with negatively charged phospholipid bilayers is essentially of electrostatic nature. Using a Scatchard approach, it is found that bilayers become saturated in Ca2+ approximately when the electroneutrality of the bilayer interface is reached. Moreover, the affinity constant was reduced by the increase of the ionic strength of the aqueous buffer. It was found that the intrinsic binding constant of Ca2+ to membranes containing 30 and 50 mol% of POPG was about 11 mM-1, in a MES buffer containing 10 mM NaCl, at pH 5.6.

Calcium↗
PubMed2006

Chemical force titrations of antigen- and antibody-modified poly(methylmethacrylate).

Poly(methylmethacrylate) (PMMA) is a versatile polymer that displays desirable properties for development of cheap and disposable microfluidic devices for sensing biomolecular interactions. Atomic force microscopy (AFM) and chemical force titrations were used to determine the efficacy of surface modifications made to accommodate protein-substrate linkage. AFM images show the effects on surface morphology of carboxylated-, amine-, hCG antigen- and anti-hCG antibody-modified PMMA substrates. Confocal microscopy was used to determine the fluorescent intensity of labeled antibody species on the PMMA substrate, confirming the success of surface antigen/antibody immobilization. Surface pK(1/2) value for carboxylic acid and amine species grafted on PMMA were determined. When carboxylic acid or amine-terminated tips were titrated against PMMA samples terminated with the hCG antigen and anti-hCG antibody, peaks appeared in the force titration curve consistent with the pI range of the antigen or antibody species. Strong adhesive forces were present at pH values above 7.0 when the antigen was present on the PMMA substrate, and these were attributed to hydrophobic interactions between the antigen and the alkane "linker" chain attaching the amine or carboxylate group to the AFM tip. Such hydrophobic interactions were not observed with the carboxylic acid or amine/antibody combinations suggesting that the surface-linked antibody was more resistant to denaturation under higher pH. The results demonstrated the feasibility of using AFM approaches for interrogating protein grafting strategies in the fabrication of PMMA-based microsystems.

Antibodies↗