Sparrow, Neil Arthur.
Continuous-flow dynamic dialysis and its application to collagen-ligand interactions.
Degree: PhD, Faculty of Science, Chemistry, 1983, Rhodes University
Studies undertaken to investigate the binding of low molecular mass analogues of polyphenolic vegetable tannins to collagen have prompted the development of a new method to investigate protein-ligand interactions. This method, the continuous-flow dynamic dialysis method (CFDD), differs from conventional dialysis procedures used for protein-ligand binding studies. In this method, the ligand concentration in the diffusate is monitored automatically at successive closely spaced time intervals while being continuously eluted from the dialysis cell. The primary data obtained by this method consists of a series of spectrophotometric absorbance measurements representing the ligand concentration in the sink compartment of a dialysis cell. This primary data is recorded by means of a data logging device onto a punched paper tape for subsequent computer processing. Two original methods are presented for analysing the primary data to extract the protein-ligand binding isotherm. The first of these is a direct analysis which relies on Fick's first law of diffusion. In this method it is necessary to establish, by means of a control experiment, a value for the ligand permeation constant. This is used in a subsequent analysis to establish a relationship between the measured rate of diffusion of the ligand from a protein-ligand mixture and the concentration of unbound ligand which is in equilibrium with the protein-ligand complex. The protein-ligand binding isotherm is obtained from parametric
equations which give the quantity of ligand bound to the protein and the concentration of unbound ligand in the sample compartment as functions of time. The second method, which is more general, utilizes the same primary data but is based on establishing a system transfer function to characterise the dialysis and eluting processes. This analysis depends on the linearity of the system and utilizes numerical laplace transforms of the primary data sets obtained from control and protein-ligand dialyses. Laplace transforms are used to effect a deconvolution of the transfer function from the primary data and yield the concentration of ligand in equilibrium with the protein-ligand complex. This procedure yields, simultaneously, both the total ligand concentration and the concentration of unbound ligand in the protein compartment of the dialysis cell. These quantities are used to establish the binding isotherm for the protein ligand system. Numerical inversion of the laplace transforms in this analysis is effected by their reduction to Fourier series. The experimental reliability of the continuous-flow dynamic dialysis method, and validity of the two analytical methods used to derive a binding isotherm from dialysis data are evaluated from studies of the binding of phenol red to bovine serum albumin (BSA) at 15⁰, 20⁰ and 25⁰ C, as well as from simulated binding curves generated by the numerical solution of the differential equations used to describe the dialysis and elution process in terms of a two-site Scatchard binding model. The method is used to…
Advisors/Committee Members: Glasser, Leslie.
Subjects/Keywords: Collagen; Ligands (Biochemistry); Ligand binding (Biochemistry); Protein-protein interactions; Tannins
to Zotero / EndNote / Reference
APA (6th Edition):
Sparrow, N. A. (1983). Continuous-flow dynamic dialysis and its application to collagen-ligand interactions. (Doctoral Dissertation). Rhodes University. Retrieved from http://hdl.handle.net/10962/d1004617
Chicago Manual of Style (16th Edition):
Sparrow, Neil Arthur. “Continuous-flow dynamic dialysis and its application to collagen-ligand interactions.” 1983. Doctoral Dissertation, Rhodes University. Accessed January 17, 2020.
MLA Handbook (7th Edition):
Sparrow, Neil Arthur. “Continuous-flow dynamic dialysis and its application to collagen-ligand interactions.” 1983. Web. 17 Jan 2020.
Sparrow NA. Continuous-flow dynamic dialysis and its application to collagen-ligand interactions. [Internet] [Doctoral dissertation]. Rhodes University; 1983. [cited 2020 Jan 17].
Available from: http://hdl.handle.net/10962/d1004617.
Council of Science Editors:
Sparrow NA. Continuous-flow dynamic dialysis and its application to collagen-ligand interactions. [Doctoral Dissertation]. Rhodes University; 1983. Available from: http://hdl.handle.net/10962/d1004617