1. The basic glutathione S-transferases from rainbow-trout liver were more stable than the acidic ones. 2. The apparent pI values of these enzymes were lowered when they were eluted from a glutathione affinity column by reduced glutathione at pH 8.85. 3. The pI effect was not a function of the high pH alone, was diminished under conditions less favourable to glutathione oxidation, and did not occur when S-hexylglutathione affinity chromatography was used instead.

A simple model of zonal gel filtration is analysed numerically to establish semiquantitatively how the capacity of a protein-ligand complex to survive gel filtration intact depends on several experimental variables and on the kinetic characteristics of the complex. The design of gel-filtration experiments aimed at detecting and partially purifying binding proteins is discussed.

1. The effect of systematic error (loss of ligand, complex or macromolecule) on three of the experimental designs by which equilibrium dialysis may be used to quantify the interaction of ligand and macromolecule is examined theoretically, and the design that is least sensitive to systematic error is identified. 2. Thirteen methods for fitting the binding isotherm to experimental data are compared by using them to analyse simulated data containing random error, and the most reliable method is identified.

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.

1. Descriptions are given of two ways for fitting non-linear equations by least-squares criteria to experimental data. One depends on solving a set of non-linear simultaneous equations, and the other on Taylor's theorem. 2. It is shown that better parameter estimates result when an equation with two or more non-linear parameters is fitted to all the sets of data simultaneously than when it is fitted to each set in turn.

Two methods are described for fitting the Michaelis-Menten equation to sets of data with a common michaelis constant but different maximum velocities. One of them uses the method of least squares, and the other is based on the direct plot of Eisenthal & Cornish-Bowden [Biochem. J. (1974) 139, 715-720].

1. The binding of cholic acid to 100000g supernatants from rat livers was investigated by equilibrium dialysis and gel-exculsion chromatography. 2. Supernatants were found to contain at least two classes of binding site for cholic acid. 3. These recptor molecules are probably proteins since incubation with proteolytic enzymes resulted in complete loss of cholic acid binding. 4. Supernatants were added to columns of Sephadex G-75, and two groups of fractions were shown to bind cholic acid. One of these contained low-affinity binding sites and the other contained both low- and high-affinity binding sites. 5. Feeding cholestyramine had no effect on cholic acid binding. 6. Increased cholic acid binding occurred after injection of phenobarbitone. There was an increase in the amount of the low-affinity component but no change in the high-affinity component. 7. The dissociation constants of the binding of cholic acid suggest that the binding proteins may be involved in bile acid transport.

The Michaelis-Menten equation was fitted to simulated data containing different sorts of error by using the three linear transformations, and the methods of S. R. Cohen [ Anal. Biochem. (1968) 22 , 549-552], R. Eisenthal & A. Cornish-Bowden [ Biochem. J. (1974) 139 , 715-120], F. de M. Merino [ Biochem. J. 143 , 93-95] and G. N. Wilkinson [ Biochem. J. (1961) 80 , 324-332). The best methods were those of Eisenthal & Cornish-Bowden (1974) and Wilkinson (1961).