Several neutral solutes, ranging in size from methanol to a tetrasaccharide, stachyose, are shown to stabilize the left-handed Z form of the methylated polynucleotide poly(dG-m5dC). The action of these solutes is consistent with an osmotic stress, that is, with their effect on water chemical potentials coupled to a difference in the number of associated water molecules between the B and Z conformations. The apparent difference in hydration between the two forms is, however, dependent on the particular solute used to probe the reaction. The effect of solutes is not consistent either with a direct binding of solute or with an indirect effect on electrostatics or ion binding through changes in the solution dielectric constant. The interplay of NaCl and neutral solute in modulating the B-Z transition suggests that salt also could be stabilizing the Z form through an osmotic stress.

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O(2) affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the "osmotic stress" method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl(-) and of DPG concentration. We find that only approximately 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with approximately 72 that bind in the presence of more than 50 mM Cl(-) or more than 15 microM DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl(-), DPG, and H(2)O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

We present data which indicate that glycine and glucose influence HbA oxygen affinity to the same extent, despite the fact that glycine increases and glucose decreases the bulk dielectric constant of the solution. Furthermore, we derive an equation linking changes in oxygen affinity to changes in differential solute and water binding to test critically the possibility of neutral solute heterotropic binding. Applied to the data, these analyses support our original interpretation that neutral solutes act indirectly on the regulation of allosteric behavior of hemoglobin by varying the chemical potential of water in solution. This leads to a displacement of the equilibrium between Hb conformational states in proportion to their differential hydration.It is well established that differential water binding occurs between distinct conformations of a protein. Hence, whether or not differential water binding contributes to the energetic of protein reactions, as some ions and metabolites do, is a key question to fully comprehend the mechanism of biological control. The role of water on allosteric regulation had been previously investigated, using Hb oxygenation as a model, by the so-called osmotic stress method (1, 2). The concept of this method is to use solutes, which are potentially excluded from the protein surface, to set the bulk water activity a w (3). Haire and Hedlund (4), using ethylene glycol, observed that at a low concentration range ethylene glycol decreases Hb oxygen affinity while at a higher concentration causes the opposite effect. They interpreted these results as reflecting preferential binding of ethylene glycol to Hb at the low solute concentration range, whereas, at high ethylene glycol concentration range, the increased O 2 binding affinity of Hb was attributed to an effect of water activity on the R to T allosteric equilibrium (4). In our former studies of the effect of water activity on Hb O 2 binding properties, we have used chemically different solutes like sucrose, stachyose, and polyethylene glycols to distinguish solute from water binding to Hb (1). Besides finding evidences for an indirect effect of solutes through water on the deoxy-to oxy-Hb conformational equilibrium, we have also determined quantitatively this solvation effect on Hb allosteric transition in terms of the differential number of water molecules bound between the two extreme conformations of Hb. By using two different model approaches, the Wyman linkage equation or the Gibbs Duhem equation, we found that 65-72 1 water molecules are linked to the binding of four oxygen molecules to human Hb under the experimental conditions used. Furthermore, this hydration change was found to be in agreement with the difference between the solvent-accessible surface areas of deoxy-and oxy-Hb computed from x-ray structures by Clothia et al. (5,6).The experimental approach and analyses originally used by us to assess and quantify the role of water on the allosteric control of Hb have been tested in several other biochemical systems (7)(8)(9)...

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