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Poly(ethylene glycol) 6000 affected many of the properties of skeletal-muscle actin. It accelerated the rate and increased the extent of actin polymerization as measured by light-scattering and sedimentation studies respectively. Moreover, intrinsic-fluorescence measurements showed that addition of poly(ethylene glycol) 6000 decreased the rate of EDTA-induced denaturation of actin monomer and increased the temperature at which irreversible conformational changes occur in actin monomer. These effects occurred without any apparent direct binding interaction and are postulated to be a consequence of the effect of excluded volume on the thermodynamic activity of actin. A relationship based on spherical geometry was formulated which described the co-volume increment that occurs upon addition of a monomer to a long linear polymer in the presence of a space-filling macromolecule. The application of this relationship to the poly(ethylene glycol) 6000-actin system was not without assumption, but it permitted quantitative estimation of the co-volume increment which proved to be of the sign and magnitude required to explain the increased extent of actin polymerization found experimentally in the presence of various concentrations of poly(ethylene glycol) 6000. It is suggested that, in vivo, excluded volume may play a role in actin-filament formation and in the maintenance of the native G-actin structure.

Bacterial alpha-amylase was shown by equilibrium and velocity-sedimentation studies to be a monomer-dimer equilibrium system in 0.10M-NaCl/0.015M-calcium acetate/0.010M-EDTA, pH7.0; an association constant of 2.4 × 10(3)M-1 is obtained. Studies of the binding of Zn2+ to alpha-amylase in 0.10M-NaCl/0.005M-calcium acetate, pH7.0, yielded binding curves that exhibit dependence on the concentration of alpha-amylase (Zn2+-free) used in the equilibrium-dialysis experiments. Results are described very satisfactorily by a reaction scheme in which Zn2+ binds exclusively to the dimer of the above monomer–dimer system with an association constant of 1.0 × 10(6)M-1. The present results refute the earlier scheme for dimer stabilization by Zn2+ in which the metal ion formed a cross-link between two non polymerizing monomer units.

A theoretical expression is derived for concentration dependence of elution volume in the gel chromatography of a non-interacting solute. Experimental results for bovine serum albumin on Sephadex G-100 are shown to be in good agreement with the predicted gel-chromatographic behaviour. The theoretical treatment of concentration dependence is extended to include a solute undergoing rapid reversible polymerization (nA in equilibrium C). Computer simulation of gel-chromatographic data for monomer-dimer systems on Sephadex G-100 is used to illustrate the deficiencies of earlier empirical approaches, and also the potential of the present treatment, of allowing for non-chemical concentration dependence in gel chromatography of polymerizing solutes.

Previously reported results of equilibrium-partition experiments on the interaction of aldolase with actin-containing filaments [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98] have been subjected to a more rigorous theoretical analysis involving consideration of the consequences of cross-linking interactions between enzyme and filament. The experimental results obtained with F-actin-tropomyosin are best described by a model with one binding site per heptameric repeat unit of filament and a value of 39000 M-1 for the site binding constant, k. Similar analyses of the influence of Ca2+ on aldolase binding to F-actin--tropomyosin--troponin substantiate the existence of two equivalent binding sites (k = 14900 M-1) for the enzyme on each repeat unit of the thin filament. The Ca2+-sensitivity of this interaction reflects either a decrease in the strength of aldolase binding to these two sites (k = 8200 M-1) or the elimination of one site.

GILBERT1developed a differential gel chromatographic technique to compare the extents of association of two different haemoglobin species. This entailed the layering of a relatively large volume of solution of one species onto a column equilibrated with an identical concentration of the other. Differences in the weight-average elution volumes of the two solutes resulted in a change of protein concentration at the junction of the two solutions, this effect being a consequence of mass conservation. We wish to explore Gilbert's suggestion1that the differential method could be adapted to study the effects of environmental factors (pH and ionic strength, I) on the state of a single solute system. The acid expansion of bovine serum albumin provides an example where a variation in pH induces a change in conformation and thus in elution volume. The effect of ionic strength on the extent of association of β-lactoglobulin A has also been studied, the change of weight-average elution volume in this instance being used to compare the association constants pertinent to two environments of different ionic strength.

A RECENT theoretical investigation of the binding of a ligand to a polymerizing protein system1extends the model of allosteric action based on isomerization2to include reversible polymerization as a basis of these effects. We discuss here the possibility of defining uniquely an allosteric system from binding data in conjunction with other experimental evidence, and we consider results on the binding of succinate to aspartate transcarbamylase3(EC 2.1.3.2) and of guanosinetriphosphate (GTP) to bovine liver glutamate dehydrogenase4(EC 1.4.1.3).

WHEN systems undergoing a rapid chemical re-equilibration between monomers and a single higher polymer are examined in the ultracentrifuge, a bimodal reaction boundary is predicted, if the degree of polymerization is greater than two1,2. The sedimentation coefficient of the monomer at infinite dilution, (SM)Omay be found by extrapolation of the weight-average values, \[smacr], determined from the rate of movement of the position corresponding to the square root of the second moment of the entire schlieren diagram at a series of total protein concentrations3, or by examining the system in an environment where aggregation is inhibited. For example, the latter procedure was used to estimate (SM)Ofor α-chymotrypsin as ∼ 2.5 S4,5. The determination of the sedimentation coefficient of the polymer, sp, however, at a particular concentration (and hence (sp)Oat infinite dilution) is difficult, for there is no defined position on the experimental schlieren pattern which corresponds to the position of the polymer at a given time. Certainly, an extrapolation of sfast, found from the rate of movement of the faster peak of the bimodal boundary, leads to serious error6,7. A value may be estimated on the basis of elementary theory8and the equations of Perrin9, by assuming a model and assigning values to (SM)Oand n2,7. The latter procedure is not satisfactory, in view of the importance of spas an essential parameter in determining: (1) the value of the equilibrium constant from s data, which could be compared with that obtained from the Gilbert theory1; (2) the theoretical concentration dependence of s values for observed peaks in sedimentation velocity experiments1,7.