The Water Effect on Allosteric Regulation of Hemoglobin Probed in Water/Glucose and Water/Glycine Solutions

Abstract

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)...

Dielectric Response of Cytoplasmic Water and Its Connection to the Vitality of Human Red Blood Cells. II. The Influence of Storage

“…54 As it is allosteric protein and as the oxygenation induces subsequent conformational transitions, 55 the ratio of bound to bulk water volumes must be steadily changing, due to binding of additional water molecules to Hb. 56,57 The implication is that less and less bound/bulk water interfaces are available. However, the same bulk water interior maintains cell integrity by regulating osmotic pressure, a problem as cellular biochemistry progresses in the interior.…”

“…As it is allosteric protein and as the oxygenation induces subsequent conformational transitions, the ratio of bound to bulk water volumes must be steadily changing, due to binding of additional water molecules to Hb. , …”

Effect of dC → d(m5C) substitutions on the folding of intramolecular triplexes with mixed TAT and C+GC base triplets

“…The activity of water was varied by increasing the concentration of ethylene glycol, an osmolyte that does not interact with DNA, from 0.5 – 2.5 M.[88] The osmolality of the ethylene glycol solutions was measured with a Model 830 UIC vapor pressure osmometer which was calibrated with standardized NaCl solutions. The osmolalities were converted into water activity using the equation lna W = −Osm/M W ,[89] where Osm is the measured solution osmolality and M W is the molality of pure water. Measurement of the differential binding of water using the osmotic stress method yields only the immobilization of structural, not electrostricted, water.…”

Thermodynamic Stability of DNA Duplexes Comprising the Simplest T → dU Substitutions

“…19 The osmolality of the latter solutions was obtained with a model 830 UIC vapor pressure osmometer, which was calibrated with standardized solutions of NaCl. These osmolalities were then converted into water activities (a w ) using the following equation: 20 ln a w = −Osm/ M w , where Osm is the solution osmolality and M w is the molality of pure water equal to 55.5 mol/kg of H 2 O.…”