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PubMed1986

An evaluation of the hydration of lysozyme by an NMR titration method.

In this study a new titration method is proposed to study the motional properties of water molecules in conjunction with globular proteins using proton NMR relaxation measurements. The method was applied to the study of the interaction of water with lysozyme and allowed identification of four water fractions-superbound water, polar-bound water, structured water and bulk water - in exchanged equilibrium. The titration demonstrated that 193 water molecules are hydrogen bonded directly to the lysozyme molecule. The combination of structured and bound water extends to 1.4 g H2O per g lysozyme and approx. two to three layers from the surface of the macromolecule. It is proposed that this structured water is related to non-isotropic water rotation in conjunction with hydrophobic patches and directly related to 'hydrophobic bonding' changes. Water amounts greater than 1.4 g H2O per g lysozyme are sufficiently distant from the macromolecule for motion to revert to that typical of water in bulk. The typical correlation times for water motion in the four fraction are: over 10(-6) s (superbound); 10(-9) s (polar bound); 10(-11) s (structured) and 10(-12) s (bulk). These results correlate well with results from other measurement techniques found in the literature.

Chemical Phenomena↗
PubMed1990

Non-enzymatic protein glycosylation: back-titration assay.

The glycosylation of albumin in vitro, as judged from the incorporation of D-[U-14C]glucose into trichloroacetic acid (TCA)-precipitable material represents a time-, temperature- and concentration-related process. It is markedly increased by NaCNBH3. A close correlation is observed between the radioactive data and the percentage of glycosylated albumin, as measured by affinity chromatography. The latter method, however, does not give information on the precise stoichiometry of the protein glycosylation. The relative extent of protein glycosylation can also be estimated by a back-titration procedure, the condensation of labelled D-glucose with the protein being inversely related to its prior degree of glycosylation. The back-titration assay was applied to plasma samples from normal and diabetic rats or human subjects and used to compare the glycosylation of albumin by distinct hexoses and hexose-phosphates. L-Glucose was found as efficient as D-glucose in causing albumin glycosylation and, hence, could conceivably be used to investigate in vivo changes in the intrinsic properties of extracellular proteins secondary to their glycosylation.

Animals↗
PubMed1983

Adaptations to hypercapnic conditions in the nutria (Myocastor coypus)--in vivo and in vitro CO2 titration curves.

Arterial blood acid-base status of unanesthetized, unrestrained nutria was studied during exposure to 5, 10 and 14.5% CO2 for 6 hr. Control values, pH = 7.426 +/- 0.037, PaCo2 = 36.5 +/- 3.1 mmHg and [HCO-3] = 24.3 +/- 2.5 mM/1 (n = 24), are within the normal range reported for other mammals. Values after 6 hr of exposure to 10% CO2 were: pH = 7.355 +/- 0.043, PaCO2 = 71.0 +/- 3.6 mmHg and [HCO-3] = 38.0 +/- 4.1 mM/l (n = 5). Arterial blood buffer slopes, obtained from the in vitro titration curve, did not show any pattern of adaptation to hypercapnia. Whole body buffer slopes, calculated from the in vivo CO2 titration curve, showed significantly higher values for the nutria than for the rat, dog and man, under comparable conditions [beta(delta HCO-3/delta pH)] = 57.0 slykes for nutria, 32.6 for rat and 11.8 for man. delta H+/delta PaCO2 = 0.38. mM/l/mmHg for nutria, 0.55 for rat and 0.76 for man. The results suggest that the nutria possesses an efficient metabolic mechanism for regulation of pH level during exposure to hypercapnic conditions.

Acid-Base Equilibrium↗
PubMed1980

The development of an equation for the potentiometric titration of linear polyelectrolytes using a site model.

An equation for the potentiometric titration of linear polyelectrolytes is derived by considering neighbor interactions of ionizable groups and the association equilibria between ionized groups and counterions. The first, second, ... and lth neighbor interactions are directly introduced into the equation, and additional neighbor interactions together with interactions between the ionizable groups and simple salt ions are taken into account as perturbed electrostatic potentials. It is observed that the approximation which includes the second or third neighbor interaction is sufficient for description of the potentiometric titration. Good agreement between predictions of the present theory and experiment is obtained for isotactic and syndiotactic poly(acrylic acids).

Journal Article↗
PubMed1981

Applicability of the potentiometric titration method to the analysis of the expansion process of bovine plasma albumin in acidic solutions.

To study the expansion process of bovine plasma albumin in acidic solutions, observed potentiometric titration curves at three different ionic strengths were compared with theoretical curves, using the radii of the protein determined by small angle X-ray scattering (SAXS). From the comparison, it was concluded that the expansion is completed via two different transitions and that the conformation of the protein before the first transition is stable and common at all ionic strengths, whereas the form of the protein becomes a more swollen and unstable one after the first transition. Moreover, the charge-independent part of the standard free energy change, delta G0, in the first transition was estimated from the potentiometric titration curves. The numerical value of delta G0 is 2350 +/- 50 cal/mol, which is very small compared with the corresponding one for ordinary biopolymers.

Animals↗
PubMed1988

Stability of the folded-chain beta-structure of a homopolypeptide based on time-resolved potentiometric titrations.

The standard free energy change of the unimolecular conversion between the folded-chain beta-structure and random coil of uncharged poly(S-carboxymethyl-L-cysteine) was evaluated from the potentiometric titration curves extrapolated to zero time to reduce the effect of aggregation which occurred at slower rates than chain folding for most degrees of ionization. To reduce the remaining contribution from the aggregation, the results obtained at finite concentrations were further extrapolated to zero polymer concentration. A value of -(400 +/- 100) cal/mol was obtained for a sample of chain length 630. From the titration curves at the aggregation equilibrium, the total free energy change of the whole solution associated with the same conversion was determined. The contribution made by aggregation to the stability was determined from the difference between these two quantities, which turned out to be comparable with that from the unimolecular conversion.

Drug Stability↗
PubMed1991

Interfacial thermodynamics of protein adsorption, ion co-adsorption and ion binding in solution. I. Phenomenological linkage relations for ion exchange in lysozyme chromatography and titration in solution.

In this paper we discuss the thermodynamics of ion binding in solution, protein adsorption and ion co-adsorption. The emphasis is on charge regulation effects. To this end, we introduce phenomenological linkage relations from which the ion binding can be calculated from the electrolyte dependency of proton titration curves and the co-adsorption from the electrolyte dependency of protein adsorption isotherms. The linkage relations are derived from classical interfacial thermodynamics, and thus offer an alternative approach as compared to the mass balance equations which are currently used in biotechnology, and Record et al.'s 1978 analysis of Wyman's Binding Polynomial for protein interactions. The co-adsorption theory is an extension of our previous analysis of ion binding in solution, which we include here for comparison of the ion co-adsorption with the ion binding in solution. The theory is applied to the chromatography of lysozyme on the strong cation exchanger 'mono S' and to the proton titration of lysozyme in solution. In the accompanying Part 2 of this paper the results are interpreted with a simple model.

Journal Article↗
PubMed1977

Potentiometric titration behavior of polyaspartic acid prepared by thermal polycondensation.

The dissociation behavior of polyaspartic acid prepared by thermal polycondensation was studied by potentiometric titrations at various ionic strengths. From the analysis of the titration curves, the ratio of alpha- and beta-linkaged aspartyl residues was estimated at about 7:3 and the intrinsic dissociation constants of alpha- and beta-carboxyl groups are 3.25 and 4.35, respectively.

Aspartic Acid↗
PubMed1988

The determination of water in erythromycin by Karl Fischer titration.

Important interlaboratory variation was obtained for the water content of erythromycin samples as determined by the Karl Fischer method. It is demonstrated that the variation is related to the type of reagents used. In poorly buffered systems erythromycin enol ether and water are formed by acid degradation of erythromycin. However, when an appropriate solvent is used, accurate titration of water in erythromycin is possible. A 10% m/v solution of imidazole in methanol is preferred to pyridine or a mixture of pyridine and methanol for it has a good buffer capacity, it lacks the unpleasant odour of pyridine and it allows a high titration speed.

Journal Article↗
PubMed1989

Determination of sulphated glycosaminoglycans by automated potentiometric titration with simple coated-wire electrodes.

A method for the determination of sulphated glycosaminoglycans is based on their precipitation with (1-ethoxycarbonyl)pentadecyltrimethylammonium bromide (Septonex), the excess of which is back-titrated with sodium tetraphenylborate. The titration is monitored by a simple coated-wire ion-selective electrode with a plasticized poly(vinyl chloride) membrane on aluminium wire. Under certain conditions the results are almost independent of the relative molecular mass of glycosaminoglycans. The method has been applied to the determination of the active ingredient in the pharmaceutical preparation, heparon injection.

Aluminum↗
PubMed1991

High dose titration of calcium channel blocking agents for primary pulmonary hypertension: guidelines for short-term drug testing.

Forty-seven patients with primary pulmonary hypertension were evaluated with a dose titration protocol utilizing nifedipine (20 mg orally) or diltiazem (60 mg orally) given every hour until maximal effectiveness was achieved. Of the patients tested, 15 (32%) had a greater than 20% reduction in pulmonary artery pressure (mean 36.2 +/- 8%, p less than 0.01) and pulmonary vascular resistance (mean 50.2 +/- 7%, p less than 0.01) (pressure responders). Nineteen (40%) had a greater than 20% reduction in pulmonary vascular resistance (mean 25.2 +/- 12%, p less than 0.01), with less than a 20% decrease in pulmonary artery pressure (resistance responders). Ten had no significant change in pulmonary artery pressure or pulmonary vascular resistance (nonresponders), and three were unable to tolerate the calcium channel blocking agents. No hemodynamic profile allowed prediction of the type of response to these agents. No mortality or serious morbidity was associated with the drug testing. These findings indicate that calcium channel blockers, when titrated to maximally effective doses, may cause substantial reductions in pulmonary artery pressure and pulmonary vascular resistance in patients with primary pulmonary hypertension. Testing with hemodynamic monitoring is necessary to ascertain which patients will respond. Patients with primary pulmonary hypertension are able to tolerate short-term administration of high doses of calcium channel blockers.

Adult↗
PubMedDate not supplied

Influence of self-titration on the relationship between ethanol dose and chronic tissue toxicities: theoretical considerations.

Ethanol "titration" is offered as a possible explanation for the poor correlation of typical quantity-frequency measures of alcohol consumption with the extent of neuropsychological and other performance deficits in detoxified alcoholics. This view asserts that there are large variations in the susceptibility of individuals to alcohol toxicities and that individual alcoholics may regulate their ethanol consumption in accordance with their individual susceptibility, such that usual doses and their effects fall within personally acceptable limits. Thus, the dose chosen for chronic administration is determined, in part, by the biological impact of the ethanol ingested during drinking episodes. Owing to individual differences in susceptibilities to toxic effects, this titration behavior may result in a situation in which two- or even threefold differences in alcohol intake are associated with the same levels of toxicity or performance deficits in a group of alcoholics. Under these conditions, a graded dose-response relationship cannot be expected.

Alcoholism↗
PubMed1996

Day-to-day titration to initiate transdermal fentanyl in patients with cancer pain: short- and long-term experiences in a prospective study of 39 patients.

Initial dose finding in patients with cancer pain who are started on TTS fentanyl (Duragesic, TTS-F) is often unsatisfactory with currently recommended doses and intervals. Acknowledging that studies reveal a "psuedo steady state" 15 to 20 hr after application of TTS-F, we prospectively investigated an increased initial dose and day-to-day titration of TTS-F in 39 (evaluable) patients with uncontrolled cancer pain. Significant pain reduction (P = 0.001) was seen after 24 hr, and satisfactory analgesia was achieved within 48 h and maintained for the rest of the study. Significant increases in TTS-F were necessary during weeks 1 through 4 to maintain pain control. Forty-nine percent of the patients needed one or more early dose increases. Only one patient had side effects partially due to the specific properties of the TTS. Other side effects seemed to be less common compared with usual morphine treatment. TTS-F can be titrated effectively and safely on a day-to-day basis with an increased initial dose and adequate patient monitoring, thus avoiding more complicated approaches. TTS-F seemed to induce less constipation than might be expected.

Administration, Cutaneous↗
PubMedDate not supplied

Response force titration for the assessment of the neuromuscular toxicity of 2,5-hexanedione in rats.

A silent, non-moving glass lever combined with strain gauges and mounted in an operant chamber measured the vertical isometric force exerted by the forepaw of rats. Every weekday the rats were subjected to a force titration schedule consisting of a sequence of discrete trials signalled by a lever light. The required force, above which a water reinforcement is delivered, was regulated by a generalized bisection algorithm. A stable force level which the rat was able to attain at 50% of the trials was quickly reached in each session by this algorithm. This technique was used to measure the neuromuscular performance decrement due to repeated 2,5-hexanedione treatment (250 and 500 mg/kg/day per os). The results were compared with fore- and hindlimb grip strength measurements on the same animals. This experiment showed that the force titration technique is able to distinguish between motivational and true force decrements, a distinction which cannot be made by the grip strength technique.

Analysis of Variance↗
PubMed1995

Clinical efficacy of force titrated doses of diltiazem extended-release. A placebo controlled study.

Thirty patients with clinical and ambulatory essential hypertension were treated in a double-blind fashion with force titrated doses of placebo or diltiazem once daily (OD) extended-release (ER) 120, 240, 360, and 540 mg to characterize the full-dose range of the new formulation. An incremental dose-response effect was observed both in clinic and ambulatory blood pressure parameters. Doses of > or = 240 mg significantly decreased clinic diastolic blood pressure, whereas systolic blood pressure was significantly lowered by the 540-mg dose. Moreover, compared with placebo, ambulatory blood pressure was significantly decreased by the 360- and 540-mg dose levels. Trough/peak ratios for systolic and diastolic blood pressures were 50% and higher at dose levels of 240 mg and above. Adverse reactions with diltiazem OD ER were generally mild and similar to those observed with placebo. The findings of this study indicate that the most favorable effects of diltiazem OD ER were observed in response to the 360- and 540-mg dose levels. The dose escalation forced titration study design appears to be a valuable tool in obtaining rigorous dose-response data of new antihypertensive agents.

Adolescent↗
PubMed1990

In vitro tests for the measurement of veterinary clostridial toxins, toxoids and antisera. I. Titration of Clostridium septicum toxins and antitoxins in cell culture.

The assay of Clostridium septicum antitoxin currently requires the inoculation of test mixtures intravenously into mice or intradermally into guinea-pig skin. An alternative indicator system based on the use of cell cultures is described. Evidence is presented to show that the toxins detected by the in vivo and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity and that there is a close agreement between the two methods of titration. Cell culture indicators are more sensitive than their in vivo counterparts, permitting detection of substantially lower titres than is possible using in vivo indicators. It is suggested that cell culture indicators may prove useful for the titration of Cl septicum antitoxin in sera from vaccine field trials and potency tests. Cell culture methods could also be used for the potency testing of antitoxin preparations.

Animals↗
PubMed2005

Analyzing a kinetic titration series using affinity biosensors.

The classical method of measuring binding constants with affinity-based biosensors involves testing several analyte concentrations over the same ligand surface and regenerating the surface between binding cycles. Here we describe an alternative approach to collecting kinetic binding data, which we call "kinetic titration." This method involves sequentially injecting an analyte concentration series without any regeneration steps. Through a combination of simulation and experimentation, we show that this method can be as robust as the classical method of analysis. In addition, kinetic titrations can be more efficient than the conventional data collection method and allow us to fully characterize analyte binding to ligand surfaces that are difficult to regenerate.

Antibodies, Monoclonal↗
PubMed2005

An adaptable spectroelectrochemical titrator: the midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase.

Elaborations to an earlier design of an electron paramagnetic resonance (EPR) spectroelectrochemical titrator are described. While maintaining the anaerobic capabilities of the original design, a number of modifications and revisions have been introduced. The most significant modification is the use of a detachable spectral cell, making the apparatus modular and adaptable for multiple forms of spectroscopy. Additional modifications include removable reference, auxiliary, and working electrodes; modifications to facilitate sample transfer; and adaptations for operation within an anaerobic chamber. This apparatus has been used successfully in the coulometric titration of a [4Fe-4S] enzyme, as measured by EPR spectroscopy. The midpoint reduction potential for the 2+/1+ couple in the [4Fe-4S] cluster of lysine 2,3-aminomutase is -479+/-5mV, a value that falls within the range typical of ferredoxin-like iron-sulfur clusters.

Anaerobiosis↗