A. E. Russell

An automated continuous-flow dynamic dialysis technique for investigating protein-ligand binding☆

Abstract An automated, continuous-flow dynamic dialysis technique has been developed to investigate protein-ligand binding. The method depends on a comparison of the diffusion of the low molecular mass ligand, in the presence and absence of protein, through a semipermeable membrane. The ligand passes from the sample compartment of a dialysis cell into the sink compartment through which a constant flow of eluting buffer is maintained. Digitized spectrophotometric determinations of the ligand concentration in the eluting buffer at successive, equally spaced time intervals, punched onto paper tape, provide the primary data (normally about 1000 data points). A mathematical treatment of the data based on a model of the diffusion system, whereby the protein-ligand binding isotherm may be evaluated, is discussed. The validity of the method is demonstrated from studies of the binding of phenol red to bovine serum albumin (BSA) at 15, 20, and 25°C. The method yields a large number of points on the binding isotherm (usually several hundred) which, in terms of a Scatchard model, provide values for the number of binding sites on the BSA molecule and binding constants for the phenol red-BSA interaction. The results obtained are consistent with values reported in the chemical literature but which are based on much scantier data.

Binding isotherms by continuous-flow dynamic dialysis.

The classical dynamic dialysis technique for the determination of a protein-ligand binding isotherm has been modified by the introduction of a flow cell in which the dialysate on the sink side of the membrane is continuously eluted with a constant flow of eluting buffer and its ligand concentration measured. This new experimental method is termed continuous-flow dynamic dialysis (CFDD). A transfer function procedure for extracting the binding isotherm from the dialysis data is described. This is a more general technique (requiring only a verifiable assumption of linearity) than that previously used, in which the system was modelled using Ficks first law and which relied on the establishment of quasi-steady state conditions across the membrane. The present analysis uses the Laplace transform to effect deconvolution of the impulse response function of the cell from the dialysis data and, using a Fourier series approach, directly yields numerical data representing the free ligand concentration in equilibrium with the protein-ligand complex. The protein-ligand binding isotherm is obtained in parametric form, with time as the parameter.

Effect of polyethylene glycols on the mobility of (±)-catechin on cellulose

Summary The effect of polyethylene glycols of increasing molecular weight in the aqueous developing solution on the affinity of (±)-catechin for cellulose has been investigated by paper chromatography. The mobility of catechin increased with both concentration and degree of polymerization of polyethylene glycol and an empirical expression was found to describe closely the mobility variation, of the form A n = A 1 n b where An and A1 are the respective activity functions for polymer and monomer (expressed as the molar change in RM at infinite dilution), n is the number of repeat structural units and b is a characteristic constant for the series. The form of this relationship is considered to be consistent with a model involving multipoint competitive hydrogen bonding between polyethylene glycols and cellulose. On this basis, polymer binding can be expected to increase rapidly with the number of active sites present, accounting for the displacement and resultant rapid increase in catechin mobility with degree of polymerization.

The effect of solvent composition on the mobility of (±)-catechin on cellulose

Abstract Organic solvents were used as a means of investigating the non-covalent interactions that affect the mobility of (±)-catechin on cellulose. The effect on catechin mobility of changing the solvent-water ratios is interpreted in terms of the polar bonding capacities of the solvent mixtures and the influence of water ‘structure’ on these capacities.

Effect of polyethylene glycols on the mobility of the polyphenolic constituents of wattle extract on cellulose

Abstract In a previous study, the effects of polyethylene glycols of increasing molecular weight in aqueous solution on the affinity of catechin on cellulose have been investigated by paper chromatography. In this paper, similar effects of the polyethylene glycol series on the affinity of the polyphenolic constituents of wattle extract on paper have been investigated. As for catechin, the migration of the more mobile constituents increased with concentration and, in particular, with the degree of polymerization of the various glycols, conforming to the empirical expression where An and A1 are the activity functions for polymer and monomer, respectively (expressed as the molar change in RM at infinite dilution), n is the number of repeat structural units in the molecule and b is a characteristic constant for the series. The rapid increase in mobility with polar group content of the polymers implicit in the above relationship is considered to be consistent with a mechanism involving cooperative hydrogen bonding of these groups to complementary sites on cellulose, resulting in competitive displacement of bound tannins.