Rapid equilibrium dialysis

Waters, N.J., Jones, R., Williams, G. and Sohal, B. (2008) Validation of a rapid equilibrium dialysis approach for the... 

Protein binding Rapid equilibrium dialysis < 98.5 % bound [23]... 

The techniques used include equilibrium dialysis [11] and fluorescence procedures [12]. Another method is based on the observation that the rate of binding of a-bungarotoxin (aBgt) to the receptor is affected by cholinergic ligands [13] and permits rapid estimation of the receptor affinities of a wide spectrum of nonradioactive, nondialyzable drugs. 

Another approach employed to establish the occurrence of a density nversion between the two solutions subsequent to boundary formation involves dialysis between the two solutions s0>. The dialysis membrane is impermeable to the polymer solutes but permeable to the micromolecular solvent, H20. Transfer of water across the membrane occurs until osmotic equilibrium involving equalization of water activity across the membrane is attained. Solutions equilibrated by dialysis would only undergo macroscopic density inversion at dextran concentrations above the critical concentration required for the rapid transport of PVP 36 0 50). The major difference between this type of experiment and that performed in free diffusion is that in the former only the effect of the specific solvent transport is seen which is equivalent to a density inversion occurring with respect to a membrane-fixed or solute-fixed frame of reference. Such restrictions are not imposed on free diffusion where equilibration involves transport of all components in a volume-fixed frame of reference. The solvent flow is governed specifically by the flow of the polymer solutes as described by Eq. (3) which, on rearrangement, gives... 

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). 

L Irreversible inactivation. Inactivation by affinity labels leads to irreversible covalent bond formation between the enzyme and the inhibitor. Unlike the complex between and enzyme and a rapid, reversible inhibitor, the covalent enzyme-inhibitor complex is no longer in equilibrium with free enzyme and inhibitor. Therefore, exhaustive dialysis or gel filtration of the covalent enzyme-inhibitor complex cannot lead to the recovery of free, active enzyme. However, such experiments do not allow distinction among tight-binding, noncovalent inhibitors, affinity labels, and mechanism-based inactivators. 

The binding forces are electrostatic attractions between sites with opposite charges such as -NH3 carried by a lysine residue and a carboxylate, van der Waals forces, and hydrogen bonds. It is predicted that the first two types of bonds, which increase rapidly as the distances decrease, will be all the more efficient as the complementarity is better. The expression hydrophobic interactions is used because an exact fit drives away water molecules present close to the hydrophobic residues such as valine, leucine, isoleucine, etc. This concept, developed further in Chapter 11, implies that a decrease in surface contact with water is in itself a stabilizing factor. All these forces are reversible. When the hapten-antibody complex in solution is introduced into a dialysis bag, only the hapten can cross over the membrane, and its elimination from the interior bag causes complete dissociation of the complex through equilibrium displacement. 

Although the large scale industrial utilisation of ion-exchange membranes began only 20 years ago, their principle has been known for about 100 years [1]. Beginning with the work of Ostwald in 1890, who discovered the existence of a "membrane potential" at the boundary between a semipermeable membrane and the solution as a consequence of the difference in concentration. In 1911 Donnan [2] developed a mathematical equation describing the concentration equilibrium. The first use of electrodialysis in mass separation dates back to 1903, when Morse and Pierce [3] introduced electrodes into two solutions separated by a dialysis membrane and found that electrolytes could be removed more rapidly from a feed solution with the application of an electrical potential. 

The in-vitro drug release kinetic profile of clofibride from a nanoemulsion was determined using two different kinetic techniques the bulk equilibrium reverse dialysis sac technique, and the centrifugal ultrafiltration technique at low pressure. The former technique was shown to be inadequate for in-vitro kinetic comparison purposes as a result of drug diffusion limitations through the dialysis membrane. The latter technique yielded rapid in-vitro release profiles of clofibride from the nanoemulsion under perfect sink conditions. The kinetic results clearly exclude the use of a nanoemulsion as a colloidal controlled release delivery systems for any administration route where perfect sink conditions should prevail. ... 

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