Stanley Ainsworth

The binding of aromatic sulphonic acids to bovine serum albumin

Abstract The partitioning of several naphthylamine sulphonate derivatives between water and solutions of dodecylamine in hexane and between water and detergent micelles was studied as an aid to understanding the binding of these dyes to bovine serum albumin. The data obtained indicate that the distribution of the dyes between the two phases depends both on non-polar and electrostatic interactions. A similar dependence was found in the binding of these dyes to bovine serum albumin. Further studies with l -thyroxine and Trypan blue showed that these molecules are bound to bovine serum albumin at the sites where naphthalymaine binding occurs. The binding of Trypan blue showed interaction effects and an explanation for this is suggested in terms of a competition by the functional groups of Trypan blue for a limited number of sites.

The effects that the environment exerts on the spectroscopic properties of certain dyes that are bound by bovine serum albumin.

Abstract Experiments are reported in which the polarity of the dye binding sites of bovine serum albumin was estimated by changes in the spectroscopic properties of 1-anilinonaphthalene-8-sulphonic acid, 1-dimethylaminonaphthalene-5-sulphonic acid, Rhodamine B and Phenol Blue. The polarity indicated by the first two dyes was less than that indicated by the second pair. Experiments are reported that show all the dyes are bound to the same protein sites and an explanation of the discrepancy is suggested based on the demonstration that the spectroscopic properties of the naphthalene sulphonic acids depend not only on the polarity of the solvent but also on its rigidity and its ability to solvate the dye.

A simple model for a regulatory enzyme

A simple model for a regulatory enzyme is described which leads to relationships between the initial velocity of the catalysed reaction and the varied concentration of a substrate that are of the non-inflected or sigmoidal varieties without a maximum. The model assumes that the most relevant measure of protein configuration (itself determining the kinetic behaviour of the enzyme) is the apparent association constant, αi, measured for the given fractional saturation of the ligand under investigation. It is further assumed that the original state of the protein in solution, α0, is destabilized by an increment of energy, ΔGp0, that is proportional to the fractional saturation of the enzyme by ligand so that the formation of a new configurational state, a,, can be represented by −ΔGp0 = RT ln α1α0. The rate or fractional saturation equation that can be derived from this model predicts both positive and negative cooperativity. Either equation can be transformed for linear representation, provided the maximum velocity or its equivalent maximum saturation is known, and estimates of α0 and αi (the apparent association constants at zero and complete saturation) can be obtained thereby. A procedure is also described by which an initial estimate of the maximum velocity or saturation can be improved. The model is tested by application to a range of data in the literature and it is shown to give fits to the data comparable in quality to those provided by the model of Monod, Wyman & Changeux (1965).

Allosteric properties of rabbit muscle pyruvate kinase

Abstract 1. 1. The paper reports that rabbit muscle pyruvate kinase displays kinetic properties typical of a regulatory enzyme when examined at pH 7.4. 2. 2. Time-dependent conformational changes in the enzyme are shown. 3. 3. Reasons are suggested to explain why these properties have escaped attention. 4. 4. It is suggested that the results can be explained by assuming that the substrates of the enzyme and the activator, fructose-1, 6-diphosphate, induce conformational changes in the enzyme, some of which are antagonistic in their effects.

A computer program to derive the rate equations of enzyme catalysed reactions with up to ten enzyme-containing intermediates in the reaction mechanism

The paper describes a program, designed for a desk-top computer, which can be used to derive the rate equations of enzyme catalysed reactions with up to ten enzyme-containing intermediates included in the mechanism. The program allows the rate equation to be presented in simplified forms of practical use and in a variety of formats.

Reactions of partially oxidised hemoglobin solutions II. A matrix rank analysis of the initial rates of binding carbon monoxide

Abstract Data are presented for the initial rates of combination of CO with partially oxidised solutions of a sheep hemoglobin of the B type, the initial rates being measured as a function of the fraction of the pigment initially present as hemoglobin and the pH of the solution. The experimental data are subjected to a matrix rank analysis to ascertain the number of independent constants required to describe the combination reactions. The evidence suggests that three such constants are required.

The exponential model for a regulatory enzyme: Its extension to describe the binding of two ligands

Abstract The exponential model for a regulatory enzyme (Ainsworth, 1977 a ) is extended to deal explicitly with the presence in solution of a second ligand. This is achieved by introducing exponential interaction coefficients which respectively describe how the affinity of the free and bound forms of the protein for the ligand depend on its fractional saturation by the second ligand. The basic equations, so derived, are applied to binding experiments where the ligands bind independently or competitively and to rate experiments where the ligands represent two substrates or one substrate and a modifier which may be either competitive or non-competitive in type. The conditions required to display linkage between the binding of the two ligands are established and it is also shown that rate data may display a maximum as one ligand concentration is varied at a fixed concentration of the other. The equations that are derived are tested by application to experimental data and the conditions that have to be met to justify such an application are discussed.

The mechanistic interpretation of initial velocity and product inhibition data arising from enzyme catalysed reactions whose catalytic cycle is unbranched

Abstract A procedure is described which determines the mechanisms of unbranched enzyme catalysed reactions from a knowledge of the experimentally determined kinetic constants. The basis of complementarity in enzymic mechanisms is also discussed.

Reactions of partially oxidised hemoglobin solutions I. Separation of intermediate compounds by CM-sephadex chromatography

Abstract The separation of hemoglobin derivatives containing two iron atoms in the ferric state is described. Using a sheep hemoglobin of the B type, it was found that a pH-gradient elution of partially oxidised oxy- or carboxyhemoglobin from CM-Sephadex produced three fractions which, in order of elution, corresponded to unoxidised, half-oxidised and wholly oxidised pigment. The same result followed if hemoglobin derivatives were mixed with methemoglobin solutions and stored for several days.

A high-speed stopped-flow apparatus

Abstract 1. 1. The paper describes a stopped-flow apparatus developed from the original Gibson and Milnes (1964) design. 2. 2. The new design is more rugged in construction than the original, incorporates simplified flow channels for the reactants and is powered automatically by air pressure. 3. 3. As a result of these modifications, a significant lowering of the dead time of the apparatus has been achieved. 4. 4. The progress of the reaction can be monitored by absorption, and fluorescence measurements. 5. 5. The apparatus can also be modified to permit measurement of circular dichroism changes (Anson & Bayley, 1974).