Coombe, RG & George, AM 1976, 'A rapid method for the purification of β-lactamase from Bacillus cereus by affinity chromatography', Analytical Biochemistry, vol. 75, no. 2, pp. 652-655.
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A procedure for the purification of β-lactamase from Bacillus cereus in a single chromatographic step is described. The enzyme is isolated from the crude culture supernatant by affinity chromatography. An inhibitor, methicillin, was immobilised by covalent attachment to the insoluble column gel, Sepharose. The enzyme was adsorbed to the column ligand from the crude supernatant and was subsequently released by increasing the ionic strength of the eluting buffer. In this way the enzyme was selectively isolated from other proteins in the crude supernatant. About 98% of the original β-lactamase activity was recovered in the purified enzyme fraction. © 1976.
JEFFREY, PD, MILTHORPE, BK & NICHOL, LW 1976, 'POLYMERIZATION PATTERN OF INSULIN AT PH 7.0', BIOCHEMISTRY, vol. 15, no. 21, pp. 4660-4665.
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Sedimentation equilibrium results, obtained with bovine zinc-free insulin (with and without a component of proinsulin) at pH 7.0/0.2, 25 °C, and up to a total concentration of 0.8 g/1., are shown to be consistent with three different polymerization patterns, all involving an isodesmic indefinite self-association of specified oligomeric species. The analysis procedure, based on closed solutions formed by summing infinite series, yields for each pattern a set of equilibrium constants. It is shown that a distinction between the possible patterns can be made by analyzing sedimentation equilibrium results obtained in a higher total concentration range (up to 4 g/1.) with insulin freed of zinc and proinsulin, account being taken of the composition dependence of activity coefficients. The favored pattern, which differs from that previously reported in the literature, involves the dimerization of monomeric insulin (mol wt 5734), governed by a dimerization constant of 11 × 104 M-1 and the isodesmic indefinite self-association of the dimer, described by an association constant of 1.7 ×104 M-1. This polymerization pattern is also shown to be consistent with the reaction boundary observed in sedimentation velocity experiments. © 1976, American Chemical Society. All rights reserved.
NICHOL, LW, JEFFREY, PD & MILTHORPE, BK 1976, 'ANALYSIS OF SEDIMENTATION EQUILIBRIUM RESULTS OBTAINED WITH INDEFINITELY SELF-ASSOCIATING SYSTEMS USING A PROCEDURE BASED ON LAPLACE TRANSFORMATION', JOURNAL OF PHYSICAL CHEMISTRY, vol. 80, no. 10, pp. 1071-1075.
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Equations are developed in closed form which permit the simulation of the distribution of total concentration vs. radial distance obtainable in the sedimentation equilibrium of a system of specified initial concentration undergoing indefinite self-association. Simulated distributions obtained with a variety of systems involving one or more equilibrium constants are used to test an analysis procedure which fits the distribution to a function capable of inverse Laplace transformation and leads to a specification of the relative amounts of the species in the cell as a function of their molecular weights. It is shown that such results may be related to the equilibrium concentrations of oligomeric forms at each radial distance, thereby permitting successive equilibrium constants appropriate to the indefinite self-association to be estimated.
NICHOL, LW, JEFFREY, PD & MILTHORPE, BK 1976, 'SEDIMENTATION EQUILIBRIUM OF HETEROGENEOUSLY ASSOCIATING SYSTEMS AND MIXTURES OF NON-INTERACTING SOLUTES - ANALYSIS WITHOUT DETERMINATION OF MOLECULAR-WEIGHT AVERAGES', BIOPHYSICAL CHEMISTRY, vol. 4, no. 3, pp. 259-267.
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Sedimentation equilibrium is first considered of a system in which a ligand of any size binds to an acceptor at p sites, the experimental result, obtained with either interference or absorption optics, being a distribution of total solute concentration as a function of radial distance. Theory illustrated by a numerical example, is presented which shows that this distribution may be analysed to give the activity of the unbound ligand as a function of total weight concentration. It is shown that this information may be used together with conservation of mass equations written in terms of the initial mixing composition to evaluate the equilibrium constant(s) relevant to the system. Correlation with composition evaluation by use of absorption optics (when possible) is also discussed. The procedure does not involve solution of simultaneous equations which are sums of exponentials nor differentiation of experimental results to obtain apparent weight-average molecular weights. It is general in that it leads to the evaluation of the activity of the species characterized by the smallest M(l-Image ?) product and, accordingly, is shown to be useful in the analysis of non-interacting as well as of interacting systems.
