Oxygen binding in cyanmet hybrid and normal hemoglobins: applicability of sequential and two-state concerted models.

The cyanmet hybrid hemoglobins alpha(2)beta(+CN)(2) and alpha(+CN)(2)beta(2) are widely held to be similar or equivalent in structure and subunit interactions to the partially oxygen-liganded species alpha(2)(beta * O(2))(2) and (alpha * O(2))(2)beta(2), respectively. An analysis of precise data on oxygen binding to the cyanmet hybrids and normal hemoglobin shows that if this is the case, then cooperative ligand binding in hemoglobin is more properly described by some model of the sequential type than by any twostate concerted model.

The Hill plot and the energy of interaction in hemoglobin.

The Hill plot does not independently yield the average free energy of interaction per binding site, as has been proposed by Wyman, but rather the difference between the free energies of interaction on binding the first and last ligands. It is shown that additional data (or assumptions) and a model for cooperative behavior are required to obtain the average free energy of interaction.

New developments in the study of biomolecular associations via sedimentation equilibrium.

The measurement and analysis of sedimentation equilibrium provide one of the most powerful techniques for quantitative characterization of reversible and irreversible macromolecular associations in solution. The use of this technique by nonspecialists has been greatly helped in recent years by the development of new instrumentation, new types of experiments and new PC-based software for computer-aided analysis of experimental results.

Kinetic analysis of biosensor data: elementary tests for self-consistency.

The validity of the most common kinetic interpretation of biosensor data can be quickly assessed with the aid of two simple tests for self-consistency, requiring only back-of-the-envelope calculations. A search of the recent literature reveals that many published results fail these tests qualitatively.

Protein folding: Thickening the broth.

Recent results support the notion that macromolecular 'crowding' enhances protein aggregation, at the expense of correct folding. The results can be rationalised in terms of kinetic competition between distinct processes, taking into account the relative influence of crowding on each process.

Implications of macromolecular crowding for protein assembly.

Recent studies have led to increased appreciation of the influence of excluded volume in solutions of high total macromolecular content ('macromolecular crowding') upon the various classes of reaction that lead to the assembly of proteins and protein complexes. In general, crowding is expected to stabilize native protein structure relative to less compact non-native structures and to favor the formation of functional complexes of native proteins. Under certain pathological conditions, 'overcrowding' may enhance the formation of nonfunctional aggregates of non-native protein (e.g. amyloid and inclusion bodies).

On the interpretation of binding isotherms in complex biological systems. Apparent homogeneity of some heterogeneous systems.

A simple treatment of the effect of site heterogeneity upon binding isotherms is presented, which is applicable to the analysis of data obtained from measurements of hormone, drug, or lectin binding to membranes and cell surfaces. Using this treatment, isotherms corresponding to various distributions of binding constants have been fitted to examples of experimental binding data ordinarily interpreted in the context of a homogeneous binding site model. It is found that these data do not permit one to exclude the alternate possibility of a broad distribution of the binding constant K. If a homogeneous binding site model can be satisfactorily fitted to the data, it is probable that the value of K obtained by this procedure is equal or nearly equal to the number average value of K in the actual (unknown) distribution.

Evidence for protein self-association induced by excluded volume. Myoglobin in the presence of globular proteins.

The fluorescence polarization of fluorescent derivatives of hemoglobin and myoglobin was measured as a function of the concentration of added polymers (PEG-6 000, PEG-20 000) and globular proteins (lysozyme, ribonuclease A, beta-lactoglobulin). The results indicated that the effective size and shape of 1-anilino-9-naphthalene sulfonate myoglobin are unaltered in the presence of up to 25 g/dl poly(ethylene glycol), whereas they are significantly altered in the presence of comparable concentrations of other proteins. The results are consistent with the hypothesis that in the presence of high concentrations of added protein, 1-anilino-9-naphthalene sulfonate myoglobin self-associates to form a dimer similar in size and shape to 1-anilino-9-naphthalene sulfonate hemoglobin.

Effects of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells.

Dog red cell membranes contain two distinct volume-sensitive transporters: swelling-activated K-Cl cotransport and shrinkage-activated Na/H exchange. Cells were prepared with intracellular salt concentration and weight percentage of cell water (%cw) varied independently by transient permeabilization of the cell membrane to cations. The dependence of transporter-mediated Na and K influxes upon %cw and upon extracellular salt concentration (c(ext)) was measured in cells so prepared. It was found that the critical value of %cw at which transporters are activated, called the set point, is similar for the two transporters, and that the set points for the two transporters decrease similarly with increasing extracellular salt concentration. These findings suggest a common mechanism of regulation of these two transporters. Cellular Na, K, and Cl concentrations were measured as functions of %cw and c(ext). Using these data together with data from the literature for other solute concentrations, empirical expressions were developed to describe the dependence of the intracellular concentrations of all significant small molecule electrolytes, and therefore the intracellular ionic strength, upon %cw and c(ext). A mechanistic model for the dependence of the set point of an individual transporter upon intracellular ionic strength is proposed. According to this model, the set point represents a critical extent of association between the transporter and a postulated soluble regulatory protein, called regulator. Model functions are presented for the calculation of the thermodynamic activity of regulator, and hence extent of regulator-transporter association, as a function of total intracellular protein concentration (or %cw) and ionic strength. The experimentally observed dependence of set point %cw on c(ext) are simulated using these functions and the empirical expressions described above, together with reasonable but not uniquely determined values of model parameters.

The effect of self-association on the interaction of the Escherichia coli regulatory protein TyrR with DNA.

The interaction of the Escherichia coli regulatory protein TyrR, with a 42 bp oligonucleotide (42A/42B) containing a centrally located recognition sequence (TyrR box), was examined by analytical ultracentrifugation. The stoichiometry of the binding of oligonucleotide to dimeric TyrR was determined by equilibrium centrifugation of a mixture of fluorescein-5-isothiocyanate-labelled 42A/42B (F-42A/42B) in the presence of an eightfold molar excess of TyrR. The molecular mass (M) of the labelled oligonucleotide was estimated as 148,000, indicating a 1:1 complex composed of oligonucleotide (M = 27,000) and TyrR dimer (M = 113,000). The association constant (Ko,d = 2.8(+/- 0.1) x 10(6) M-1) was determined by a global analysis of sedimentation data, collected at multiple wavelengths between 230 and 285 nm. The presence of 30 microM ATP gamma S enhanced the affinity of TyrR for DNA approximately 3.5-fold, (Ko,d = 9.9(+/- 0.3) x 10(6) M-1). The effect of dimer to hexamer self-association of TyrR on the binding of 42A/42B was also examined. Multiple wavelength sedimentation data fitted a model in which the oligonucleotide could bind to one site on the dimer (Ko,d = 9.9 x 10(6) M-1), and to either one or three sites on the hexamer (Ko,h) = 2.0(+/- 0.1) x 10(6) M-1 and 3.8(+/- 0.1) x 10(6) M-1, respectively). Competitive sedimentation equilibrium and fluorescence anisotropy titrations were performed under stoichiometric conditions to resolve the number of oligonucleotide binding sites per hexamer. In these experiments, 42A/42B was used as a competitor to displace F-42A/42B from the hexamer, which was found to bind the 42mer with a 1:1 stoichiometry. Our data support a model in which ATP increases the affinity of TyrR for the DNA recognition sequence, and tyrosine induced self-association of TyrR generates a hexameric species with a single binding site for the 42A/42B oligonucleotide.

Influence of excluded volume upon macromolecular structure and associations in 'crowded' media.

Results of experimental studies published since the last major review of excluded volume effects in biopolymer solutions in 1993 add to our appreciation of the scope and magnitude of such effects. Recent theoretical studies have improved incrementally our ability to understand and model excluded volume effects in simple model systems.

Quantitative characterization of reversible macromolecular associations via sedimentation equilibrium: an introduction.

The measurement and analysis of sedimentation equilibrium provides one of the most powerful and widely applicable methods for the characterization of reversible associations of macromolecules in solution. Recent developments in instrumentation, experimental design, and data analysis have substantially broadened the range of systems to which this technique may be applied, simplified its application, and reduced the cost of acquiring analytical capability.

Acceleration of fibrin gel formation by unrelated proteins.

Addition of gamma-globulin, serum albumin, hemoglobin, or ovalbumin in concentrations of 1-10 g/dl to solutions of purified fibrinogen results in a substantial (up to six-fold) decrease in the lag time preceding appearance of a firm fibrin gel following addition of thrombin at 24 degrees C. The effect does not appear to be due to a protein-induced enhancement in the enzymatic activity of thrombin, nor does it appear to be due to the co-condensation of the added protein with fibrin/fibrinogen. It is suggested that the observed effect is primarily due to nonspecific volume exclusion arising from increased fractional occupancy of solution volume by macromolecules.

Thermodynamic nonideality and the dependence of partition coefficient upon solute concentration in exclusion chromatography. Application to self-associating and non-self-associating solutes. Application to hemoglobin.

The theory of partition of macromolecular solute between mobile and stationary phases under highly nonideal conditions is reexamined. Expressions are derived for the dependence of the partition coefficient of a non-self-associating solute, and the weight average partition coefficient of a dimerizing solute, upon the total concentration of solute in the mobile phase. These expressions are based upon a treatment of nonideality in the stationary phase which is more realistic than that proposed earlier [L.W. Nichol, R.J. Siezin and D.J. Winzor, Biophys. Chem. 9 (1979) 17]. It is found that the chromatographic data on oxyhemoglobin in the above paper may be accommodated by the present theory without invoking self-association.

Simulation of the time course of macromolecular separations in an ultracentrifuge. I. Formation of a cesium chloride density gradient at 25 degrees C.

A method is described for rapid numerical simulation of the time course of the formation of a density gradient of CsCl in an ultracentrifuge at 25 degrees C. Results of simulations compare well with those of experiments carried out in analytical and preparative ultracentrifuges.

Solubility relationships in binary mixtures of hemoglobin variants Application to the "gelationrd of sickle-cell hemoglobin.

A thermodynamic treatment of solubility in binary mixtures of hemoglobin variants is presented. It is shown that the reported dependence of the minimum gelling concentration in five binary mixtures of the variants S, C(Harlem') Korle-Bu and A may be satisfactorily accounted for using the derived solubility relations together with simple models relating structure to interaction energies in the condensed phase.

Effects of excluded surface area and adsorbate clustering on surface adsorption of proteins I. Equilibrium models.

Statistical-thermodynamic models for the equilibrium adsorption of proteins onto homogeneous, locally planar surfaces are presented. An extension of earlier work [R.C. Chatelier, A.P. Minton, Biophys. J. 71 (1996) 2367], the models presented here allow for the formation of a broadly heterogeneous population of adsorbate clusters in addition to excluded volume interactions between all adsorbate species. Calculations are carried out for three simple models for the structure of adsorbate, illustrating similarities and differences in the equilibrium properties of maximally compact clusters, minimally compact clusters and isomerizing clusters. Depending upon the strength of attractive interactions between adsorbate molecules, the resulting equilibrium isotherms may exhibit negative cooperativity, positive cooperativity, essentially no apparent cooperativity, or a mixture of positive cooperativity at low surface density and negative cooperativity at high surface density of adsorbate. The condition of apparent lack of cooperativity, which might naively be interpreted as evidence of a lack of interaction between adsorbate molecules, actually conceals a balance between attractive and repulsive interactions and extensive clustering of adsorbate.

A molecular model for the dependence of the osmotic pressure of bovine serum albumin upon concentration and pH.

Expressions derived from the effective hard particle model of Minton and Edelhoch (Biopolymers, 21 (1982) 451) account quantitatively for the combined data of Kanal et al. (Biophys. J., 66 (1994) 153) describing the osmotic pressure of bovine serum albumin as a function of protein concentration (< or = ca. 100 milligrams) and pH (3-8) in buffered 0.1 M NaCl. The best fit of the model yields a molar mass of 68360 and a pH-dependent value of the effective specific volume ranging from a minimum of -0.17 cm3/g at pH 4.6 (the isoelectric point) to maxima of 3.1 cm3/g at pH 3.0 and 2.2 cm3/g at pH 8.0. These values are shown to be consistent with the magnitude of known attractive and repulsive electrostatic interactions between proteins in solution.

Thermodynamic nonideality and the dependence of partition coefficient upon solute concentration in exclusion chromatography. II. An improved theory of equilibrium partitioning of concentrated protein solutions. Application to hemoglobin.

An improved theory for the partitioning of protein between concentrated bulk solution and solution sequestered in a porous medium is presented. The theory is based upon the assumption that the sequestered solution may be formally represented as a sum of three compartments: (1) a compartment which Is inaccessible to protein, but accessible to solvent and small molecule solutes; (2) a surface layer immediately adjacent to the pore boundary, within which protein molecules are constrained to quasi-two-dimensional motion; and (3) the remaining volume accessible to protein, within which the protein molecules behave as if in bulk solution. The dependence of the partition coefficient of hemoglobin upon protein concentration over the range 10-225 g/l, calculated using the theory presented, is found to agree quantitatively with experimental data presented previously (R.J. Siezen, L.W. Nichol and D.J. Winzor, Biophys. Chem. 14 (1981) 221) without invoking self-association of hemoglobin molecules.

Dissociation equilibria of the tryptophan synthase alpha 2 beta 2 complex in saline buffer and guanidine isothiocyanate, as studied by sedimentation equilibrium.

The dissociation equilibria of Salmonella typhimurium tryptophan synthase alpha 2 beta 2 complex were studied via centrifugation of the complex to sedimentation equilibrium in neutral saline buffers containing 0 to 137 mM guanidine isothiocyanate (GuSCN). The resulting concentration gradients were analyzed in the context of an equilibrium model for sequential dissociation of two alpha subunits from a stable beta 2 subunit. Under the conditions of these experiments, the first dissociation constant alone could be evaluated at GuSCN concentrations < or = 100 mM, and the second dissociation constant alone could be evaluated at GuSCN = 137 mM. At intermediate GuSCN, both dissociation constants were sufficiently well defined to rule out the presence of a large equilibrium cooperative effect in the stepwise dissociation of the alpha subunits.