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Protein binding studies by diafiltration Palmer, Cecily M.


A diafiltration technique was used to study drug-protein interactions. Fraction V human serum albumin and plasma and two drugs (phenylbutazone and bishydroxycoumarin) with a high affinity for these substances were used in this investigation. Preliminary experiments were carried out to check for release of foreign substances and for binding of drug to the Amicon diafiltration apparatus. A binding experiment, in the absence of drug, revealed release of a protein-like, ultraviolet absorbing substance from Fraction V human serum albumin. The most suitable method of purification for albumin was by diafiltration with Tris buffer. Binding curves for bishydroxycoumarin - human serum albumin, phenylbutazone - human serum albumin, and bishydroxycoumarin - plasma interactions were obtained. The r and r/Df [subscript omitted] values were calculated and binding parameters estimated by both graphical extrapolation and by a computer non-linear least squares fit analysis. Binding curves were not independent of human serum albumin concentration, but the cause of this effect was not fully resolved. Results showed the diafiltration technique can yield precise data, can be used over a wide macromolecule concentration range and produces a binding curve, from one experiment, over a wide range of molar binding ratios. Use of the Amicon diafiltration apparatus in desorption (washout) experiments and equilibrium or direct experiments was also investigated. Attempts were made to obtain binding data by centrifugation (ultrafiltration) and by a gel filtration technique (Sephadex G-25 batch method). These methods yielded unsatisfactory results which could not be compared with those obtained by diafiltration. This abstract represents the true contents of the thesis submitted.

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