Evaluation of equilibrium constants by affinity chromatography

Abstract

Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100-lysozyme-d-glucose system in acetate-chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m(-1) and 1.2x10(6)m(-1) are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 10(5) glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.

Oligomeric state of hypoxanthine−guanine phosphoribosyltransferase from Mycobacterium tuberculosis

“…2A) diverges from the linear calibration plot. This relative retardation is consistent with enzyme migration governed by a superimposed protein−matrix interaction on the size-exclusion process, the phenomenon responsible for the retarded elution of lysozyme from Sephadex matrices [17]. Vertical displacement of the elution volumes for the other three protein standards to account for enzyme retardation leads to the revised calibration plot (open symbols and solid line) (inset (Fig.…”

Partial Purification of Acetic Acid Esterase from Malted Barley

“…NaCl was included in the elution buffer to reduce ionic interactions between the protein and the column matrix, although it was realised this might increase hydrophobic interactions 27 . Glucose was used since it had been used in the elution buffer for the purification of lysozyme on Sephadex G-100 23 . Lysozyme gave uncharacteristically high elution volumes, leading to underestimates of its known molecular weight when glucose was omitted from the elution medium.…”

Foreword to ‘Quantitative and analytical relations in biochemistry’—a special issue in honour of Donald J. Winzor’s 80th birthday

“…The first problem tackled was the development of a set of quantitative expressions for the characterisation of all possible combinations of solute-matrix, solute-ligand and matrix-ligand interactions by quantitative affinity chromatography (Nichol et al 1974), a technique introduced by Don in conjunction with Pat Andrews (University of Reading) the year before. A solution to the problem of allowance for the effects of osmotic gel shrinkage on the characterisation of protein self-association by gel chromatography then followed (Baghurst et al 1975).…”

“…Initially, this behaviour was attributed (somewhat intuitively) to spatial considerations, which suggested that further acceptor attachment after the initial event was unlikely to occur (Nichol et al 1974). This potential paradox was resolved by quantitative solution to the problem of acceptor multivalence in its interaction with matrix-bound affinity sites Winzor et al 1982), a problem solved initially by means of Flory reacted-site probability theory but later handled using a different approach (Lollar and Winzor 2014).…”