Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin, I. P. 1968

Electrochemistry of Proteins. Protein-Water Interactions
Protein Hydration. Partial Specific Volumes of Proteins in Solution

Hydration is generally understood as the binding of Water dipoles by ions or ionic groups, as well as by dipoles or polar groups. Hydration can occur in both solid substances and solutions. Since protein molecules contain a significant number of polar groups on their surface, they are capable of binding substantial amounts of water dipoles. Thus, hydration water should be understood as the water bound to the protein macromolecule As a result of tight intermolecular interaction. The main characteristic of this water is that no electrolytes dissolve in it or penetrate into it. Since hydration water molecules are bound to the protein molecule in such a way that both partially lose their kinetic mobility, the volume of the hydrated molecule is always less than the sum of the volumes of water and protein taken separately. In other words, hydration is always accompanied by a decrease in volume. All these properties of hydration water—strong binding to the protein, inaccessibility to electrolyte ions, and volume reduction upon hydration—form The basis of its experimental determination.

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Fig. 48. Adsorption of water vapor by dry Proteins at 25 (from Neurath and Bailey, 1956)

1 — Collagen, 2 — serum albumin, 3 — zein.

Literature data on this issue are rather contradictory, as different authors have investigated protein hydration using various Methods. One such approach is to study the binding of water by dry proteins as a function of water vapor pressure in the system. As numerous experiments have shown, this process proceeds in several stages and takes 4–5 hours to complete, i.e., to achieve the maximum absorption of water by the protein. The amount of bound water depends on the vapor pressure, and this dependence is expressed by an S-shaped curve (Fig. 48).

Examination of these curves reveals that The process of water binding by dry proteins is divided into three stages. The First stage involves the almost instantaneous binding of a certain amount of water; furthermore, this water is bound so tightly that it can be separated from the protein only at very low relative humidity values—around 20%.

Such binding strength cannot be explained solely by simple water adsorption; therefore, it is believed that this fraction of water is bound to specific polar groups and, to a lesser extent, to peptide bonds. Evidence for water binding at peptide bonds is provided by the hygroscopicity of Peptides synthesized from Amino Acids with nonpolar side chains, as well as the ability of nylon* to bind water, with the course of this process also described by an S-shaped curve. The absence of side ionic chains in the nylon molecule indicates that water binding can occur exclusively via peptide groups.

Evidence for the strong bonding of the first portion of water to proteins is the high energy of this bond and the noticeable volume reduction accompanying the binding process. Theoretical calculations have shown that the binding of water molecules by the protein at the stage corresponding to the first segment of the curve is accompanied by the release of a significant amount of energy—from 3 to 6 kcal per 1 mol of bound water. Water binding at this stage is also accompanied by a noticeable increase in density: for instance, the density of egg albumin increases from 1.2655 to 1.2855 after binding 6.15% water. The specific volume decreases accordingly from 0.792 to 0.777 ml/g. On average, this volume decrease for various proteins is approximately 0.05–0.08 ml per 1 g of dry protein.

The amount of bound water in the first section of the curve for various proteins ranges from 4 to 10 g per 100 g of dry protein, i.e., from 4 to 10%. This is significantly less than the amount required to form a continuous monolayer of water on the protein molecule surface, amounting to 1/5 of that value.

When the water vapor pressure increases from 20 to 60% relative humidity, additional water binding occurs, close in magnitude to the amount of tightly bound water. However, The energy released upon binding this water layer is significantly lower, approximately 1.3–1.5 kcal/mol, while the protein density and system volume remain unchanged. All this indicates that the second solvation layer is very loose and weakly bound to the protein macromolecule; moreover, this binding apparently does not result from direct interaction with the polar groups of the protein molecule. Finally, at even higher water vapor pressures, the amount of water bound by the protein increases sharply, reaching 40–60 g per 100 g of protein. The protein density thereby decreases, reaching, for example, 1.1280 for serum albumin after binding 56.28% water.

The Mechanism of protein hydration at relatively high water vapor pressures remains unclear at present. It has been suggested that the second and subsequent water layers are bound to the first layer of oriented molecules through dipole induction. To test this hypothesis, A number of authors estimated the amount of energy released during The formation of the first, second, third, and fourth hydration layers upon TiO₂ hydration and showed that these values are respectively 6500, 1380, 220, and 70 cal per 1 mol of bound water. These figures are in good agreement with the bond energy values of water molecules for the First and Second solvation layers (3–6 kcal/mol and 1.3–1.5 kcal/mol).

* Cytology/cytology/92.html">SCHEMATIC Structure OF nylon

..—NH— (СН₂)6 — NH—СО(CH2)6—NH—СО—

Since the last three values are lower than the thermal motion energy of the molecules, it can be assumed that only a single layer of oriented water dipoles can be retained On the surface of the protein molecule, whereas the second and subsequent layers of oriented molecules are hardly likely to form at all. Indeed, if the first stage of hydration consists in the formation of an oriented water layer and is accompanied by an increase in the degree of system order, then water addition at high vapor pressures leads to a decrease in system order and an increase in its Entropy.

Summarizing the above, we can conclude that studying the Hydration of dry proteins allows us to investigate the dynamics of the process and determine the amount of tightly bound water. However, the latter is too small to form a monolayer of oriented water molecules on The surface of the protein macromolecule. Furthermore, studying the subsequent process of water binding does not allow us to clearly determine the amounts of water that go toward completing this monolayer and forming a secondary layer of oriented water dipoles. Thus, this method does not make it possible to determine the amount of hydration water per protein molecule, let alone the thickness of its molecular hydration shell.

To resolve this issue, numerous experiments have been conducted by various researchers to study protein hydration in solutions. One of the main premises of these experiments was that a certain fraction of water in protein solutions is considered free and can be determined by various methods. Attempts were made to determine bound water per unit of protein by calculating the difference between the total amount of water and the amount of free water.

The Methods for determining these two fractions of water varied widely. For instance, in some experiments, the amount of free water was estimated from the volume increase accompanying the freezing of a protein solution. In others, this value was estimated from the solubility of various non-electrolytes (urea, glucose, etc.) in protein solutions. However, neither of these approaches could yield satisfactory results, since during freezing, part of the free water could remain in a supercooled liquid state, while upon the dissolution of non-electrolytes in protein solutions, the latter could reduce the degree of hydration of the protein molecule. A more accurate method for assessing the degree of hydration should be considered the Determination of the density of the hydrated protein molecule by measuring the sedimentation velocity in salt solutions of varying density (p) and viscosity (η). By plotting η_S against p and extrapolating its linear part to the value of p corresponding to a sedimentation velocity equal to zero, the density of the hydrated particles themselves can be determined. However, to calculate the degree of hydration, it is also necessary to know the density of the dry protein. The latter is determined by placing a weighed sample of protein into a pycnometer, which is then filled with a hydrophobic liquid (benzene, bromobenzene, etc.) of known density. By determining the suspension density p — P/V, where P is the weight of the suspension in g and V is the pycnometer volume in ml, the density of the dry protein can be calculated. Knowing both the dry and hydrated protein densities, one can estimate the degree of hydration. The hydration factors of the ß-lipoprotein and various Viruses have been determined in this way. Admittedly, the obtained values turned out to be overestimated (0.6 g of water per 1 g of protein for ß-lipoprotein), because proteins form complexes with salts in salt solutions. As a result, the found densities will refer not to the hydrated protein, but to the hydrated protein-salt complex.

Thus, determining the degree of hydration of proteins is a very difficult task. Therefore, for a number of specific purposes, it is more appropriate to consider a quantity that can be precisely expressed. Such a quantity is the partial ideal volume of the protein . This value represents the volume increase that occurs when 1 g of protein is added to a very large volume of solvent. If g₁ and g₂ are the amounts of water and protein, respectively, in 1 g of the mixture, the volume occupied by 1 g of such a solvent (the specific volume) is equal to:

where are the partial specific volumes of water and protein, respectively. The partial specific volume of a protein is equal to its partial molar volume divided by its molecular weight. The partial specific volume of a protein can be determined by varying the g₂/g₁ ratio and plotting V_sp against g₂. The slope of this curve will yield an approximate value of ; as already mentioned, for various proteins it ranges from 0.69 to 0.75.

Since the determination of the partial specific volume assumes that the volume of the protein-water mixture equals the sum of the volumes of each component, protein hydration is not accounted for here. Therefore, it is impossible to estimate the degree of hydration from the value of . Nevertheless, this value is very important for calculating sedimentation constants, the internal friction coefficient, the viscosity increment, and, finally, the volume of bound water. However, before discussing the latter quantity, it is necessary to dwell on The concepts of "apparent specific volume" and "non-solvent" volume.

It is evident from formula (75) that evaluating the specific volume takes into account the partial specific volume of not only the protein, but also the water. A somewhat less rigorous approach is based on the assumption that the partial specific volume of water remains unchanged and equal to 1. Then the above equation is significantly simplified:

The value is called the apparent specific volume.

For all proteins, it is slightly smaller than the partial specific volumes. The apparent specific volume is closely related to the so-called non-solvent volume. The latter can be determined from the protein density increment I in Buffer solutions in equilibrium with dialysates of the same salt composition. If d_S is the density of the protein solution in the buffer and d_W is the density of its "equilibrium" dialysate, then

where c is the protein concentration in g/ml. By measuring the density increments of protein solutions containing varying amounts of buffer and the densities of the dialyzates of these protein solutions, the non-solvent volume can be calculated:

This volume is the sum of the volume occupied by bound water and the apparent specific volume of the protein, i.e., it equals the volume occupied by the dissolved hydrated protein in 1 ml of solution. Hence, the volume of bound water will be equal to the difference between V_H and v_K. The amount of bound water calculated in this manner was found to be about 20–50% of the weight of various proteins. Recalling that the maximum amount of water bound by dry egg albumin is about 56 g per 100 g of protein, it becomes evident that determining tightly bound water from the difference between the non-solvent and apparent specific volumes often yields significantly overestimated data.

The X-Ray Diffraction method should arguably be considered the most accurate approach for estimating the amount of hydration water. It was first applied by Bragg and Perutz, who obtained X-ray diffraction patterns of Hemoglobin crystallized from a pure aqueous solution and a sodium iodide solution. Since sodium iodide strongly scatters X-rays, this technique made it possible to identify that fraction of the crystallization water which is bound to the protein molecule with forces so strong that it remains inaccessible to the diffusion of iodine ions. It turned out that crystallization water accounts for about 50% of the hemoglobin crystal volume. However, only 36% of this crystallization water (approximately 18% of the crystal volume) is tightly bound, i.e., hydration water. Recalculated per 100 g of protein, this amount equals 22 g. What is the thickness of the hydration shell? If the gap between macromolecular layers in the hemoglobin crystal is 16.7 Å and the crystallization water is located within these gaps, hydration water accounts for only 6.1 Å (36%) of it. Consequently, the thickness of the hydration layer for each molecule is about 3 Å, which roughly corresponds to the size of a water molecule.

Thus, hydration water should be understood as the water that is closely associated with the protein molecule, localized on its surface, and has a thickness equivalent to a monomolecular layer.