Dialysis, equilibrium

Barre J, Chamourad JM, Houin G, Tillement JP (1985) Equilibrium Dialysis, Ultrafiltration and Ultracentrifugation Compared for Determining the Plasma-Protein Binding Characteristics of Valproic Acid. Clinical Chemistry 

Boudoulas S, Lush RM, McCall NA et al. (1996) Plasma protein binding of phenylacetate and phenylbutyrate, two novel antineoplastic agents. TherDrug Monit 18 714 Christensen JH, Andreasen F, Jensen EB (1980) The binding of thiopental to serum proteins determined by ultrafiltration and equilibrium dialysis. Acta Pharmacol Toxicol 47 24-32 

Jordan KH, Bruner J, Doan M et al. (2000) Automated Plasma Protein Binding Assay Implementation on a Tecan Genesis 150 and and Zymark RapidPlate. Poster presented at the LRIG SouthEast October 25 2000 Judd RL and Pesce AJ (1982) Free drug concentrations are constant in serial fractions of plasma ultrafiltrates. Clin Chem 28 1726 

Norris RLG, Ahokas JT, Ravenscroft PJ (1982) Determination of unbound fraction of disopyramide in plasma a comparison of equilibrium dialysis, ultrafiltration through dialysis membranes and ultrafree anticonvulsant drug filters. J Pharmacol Methods 7(1) 7—14 

Robieux I, Aita P, Sorio R et al. (1997) Determination of unbound etoposide concentration in ultrafiltered plasma by high-performance liquid chromatography with fluorimet-ric detection. J Chromatogr 686 35 Schuhmacher J, Buhner K, Witt-Laido A (2000) Determination of the Free Fraction and Relative Free Fraction of Drugs Strongly Bound to Plasma Proteins. Journal of Pharmaceutical Sciences 89 8 

Three experimental factors need to be borne in mind when using equilibrium dialysis. 

It may take some time to establish equilibrium, particularly if the ligand is fairly large. This can be problematical, particularly with experiments carried out at room temperature or 37 °C if the protein is unstable. Rate dialysis, or filter methods, which are more rapid may be a satisfactory way around this problem (Colowick 1969). 

In experiments where the ligand and macromolecule are charged, the equilibrium may be affected by the Donnan potential of the charged macromolecule, which is confined to one compartment. Donnan effects can be minimised by carrying out the dialysis in sufficiently high ionic strength media, such as in the presence of 0.1 M NaCl or KC1. 

Osmotic pressure leads to volume changes in the two compartments. This means that the concentration of macromolecule may also change, making it necessary to measure the actual concentrations of both ligand and macromolecule at the end of the experiment. 

One problem with the indirect methods for ligand binding described in the previous section is that we rarely know the actual concentrations of free ligand or complex, for example. Rather, these are inferred from indirect observations. Kqiiilibriiim dial si.sis a technique that gets around this problem in some instances. 

Equilibrium dialysis methods often involve the use of radioactively labelled ligands, so that very small concentrations and very tight binding can be measured directly. 

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Equilibrium dialysis was used to measure a binding constant of 103 m 1 between 87 and DNA. They interpreted these results as being consistent with intercalation of DNA by 77, indicating that azinomycin B itself may also intercalate. 

In equilibrium dialysis of a solution of a polyanion (valence Zp negative) with molar concentration Cp against a solution of imi-imivalent electrolyte CA (C = cation, A = anion) with molar concentration Cqa it was shown that the requirement for equal chemical potentials of the salt in the polyanion (a) and diffusate ()) phases results in the following relation... 

In a typical equilibrium dialysis study of charged polysaccharides an indicator ion, L (chromate), is included in the supporting electrolyte medium (phosphate buffer, pH 6.8, I 0.08) to allow assessment of the effective net charge of the polyanions via a modified form of Eq. 31, namely. 

Figure 12. Binding of calcium ions by pectate and apple pectin, measured by equilibrium dialysis gainst citrate to buffer the concentration of free calcium ions at low levels.

This assay system developed by Chaires [136] is a new, powerful and effective tool based on the fundamental thermodynamic principle of equilibrium dialysis for the discovery of ligands that bind to nucleic acids with structural and sequence selectivity. Here, identical concentrations of various nucleic acid samples are dialysed in dispodialysers against a common ligand solution. At equilibrium, the contents of the ligand bound to each nucleic acid are determined and this is correlated directly to the ligand s specificity to a particular sequence. 

The affinity and cross-reactivity of the whole serum and Fab fragments were determined using equilibrium dialysis for the affinity determination and RIA for the cross-reactivity studies. The average intrinsic affinity constant (Ko) of the antibody (Nisonoff and Pressman 1958) changed very little throughout the... 

Wimley, W. C. and White, S. H., Quantitation of electrostatic and hydrophobic membrane interactions by equilibrium dialysis and reverse-phase HPLC, Anal. Biochem., 213, 213, 1993. 

Competition for binding to heparin (in essentially salt-free solutions) has been studied for most physiological cations, by ion-exchange,372 equilibrium dialysis,373 c.d.,365 and 23Na-n.m.r. spectroscopy.370 The following order of affinity was generally observed Na+ < K+ < Mg2+ < Ca2+. 

J5. Jaume, J. C., Mendel, C. M Frost, P. H., Greenspan, F. S and Laughton, C. W., Extremely low doses of heparin release lipase activity into plasma and can thereby cause artifactual elevations in serum free thyroxine concentration as measured by equilibrium dialysis. Thyroid 6, 79-84 (1996). 

Surks, M. I., Hupart, K. H., Pan, C., and Shapiro, L. E., Normal free thyroxine in critical non-thyroidal illnesses measured by ultrafiltration of undiluted serum and equilibrium dialysis. J. Clin. Endocrinol. Metab. 67, 1031-1039 (1988). 

The very slow dissociation rates for tight binding inhibitors offer some potential clinical advantages for such compounds, as described in detail in Chapter 6. Experimental determination of the value of k, can be quite challenging for these inhibitors. We have detailed in Chapters 5 and 6 several kinetic methods for estimating the value of the dissociation rate constant. When the value of kofS is extremely low, however, alternative methods may be required to estimate this kinetic constant. For example, equilibrium dialysis over the course of hours, or even days, may be required to achieve sufficient inhibitor release from the El complex for measurement. A significant issue with approaches like this is that the enzyme may not remain stable over the extended time course of such experiments. In some cases of extremely slow inhibitor dissociation, the limits of enzyme stability will preclude accurate determination of koff the best that one can do in these cases is to provide an upper limit on the value of this rate constant. 

Temperature 25°C, equilibrium dialysis, small unilamellar vesicles (lecithin) [381]. 

Temperature 20°C, equilibrium dialysis, small unilamellar vesicles (DOPC), 0.1 M KC1 [382]. Centrifugation method (15 min, 150,000 g), brush-border membrane vesicles [433]. 

Ottiger, C. Wunderli-Allenspach, H., Partition behavior of acids and bases in a phosphatidylcholine liposome-buffer equilibrium dialysis system, Eur. J. Pharm. Sci. 5, 223-231 (1997). 

For particulate receptor preparations (intact cells or membranes), it is usual to separate bound from free ligand by either centrifugation or filtration. (For soluble receptor preparations, equilibrium dialysis, using a semipermeable membrane, or gel filtration can be employed.)... 

Protein binding parameters B, and Kh as depicted in Eq. (44), are obtained from in vitro experiments utilizing filtration, centrifugation, and dynamic dialysis or equilibrium dialysis methods. These techniques have been reviewed elsewhere [54,55],... 

Three methods were used in this research to measure the extent of binding of organic pollutants to dissolved humic materials. They were equilibrium dialysis, solubility measurements and changes in sorption behavior in the presence of humic materials. Other authors have used solubility measurements, ultrafiltration and volatilization measurements. The methods will be described in the following paragraphs. 

Kasianowicz et al. [65] described the determination of the transport of niclosamide protons across lipid bilayer membranes by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric... 

Aldrich humic acid 9.4 mg/L DOC, RP-HPLC separation, equilibrium dialysis, Landrum et al. 1984)... 

Valproic acid Direct immersion PDMS (100) GC-FID (LOD l mg/mL) Equilibrium dialysis followed SPME Krogh et al., 1995 (7)... 

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