Chemistry and Biology of Proteins - F. Haurowitz 1953

Protein-Water Interactions
Hydration of Dissolved Proteins

Attempts to investigate the Hydration of dissolved Proteins were undertaken even before the hydration behavior of dry proteins had been thoroughly studied. These studies assumed that a portion of the Water in protein solutions exists in a free state, while the remainder is bound to the protein. Various Methods are employed to determine the amounts of free and protein-bound water. One such method is based on measuring the volume expansion observed when a solution freezes. Because ice density is significantly lower than that of water, the freezing of free water is accompanied by a distinct increase in volume, which can be measured using a dilatometer. Using this method, it was found that a specific fraction of water in protein solutions does not freeze [21]. It was suggested that this particular fraction constitutes hydration water. However, it is well known that water can easily be supercooled below its freezing point without turning into ice. This fact served as the basis for objections against the aforementioned method, since it remains unclear whether the unfrozen water is genuinely hydration water or simply supercooled water.

It has also been suggested that a portion of the protein hydration water exists in the form of "ice," which, as is well known, occupies a larger volume than unfrozen water. It is possible that the significant reduction in initial volume during the enzymatic Hydrolysis of Proteins results from the transition of ice molecules into water molecules (see Ch. XVI) [22].

Another method applied for the same purpose is based on determining the solubility of urea, glucose, and other non-electrolytes in protein solutions. It was hypothesized that hydration water bound to proteins is incapable of functioning as a solvent for other solutes. The solubility of added non-electrolytes can be determined through chemical analysis using cryoscopy or by measuring vapor pressure. However, this non-solvent volume method has several serious drawbacks. One of them stems from the Introduction of a foreign substance into the system, which may compete with the protein for water and thereby reduce the initial degree of hydration. Another stems from our inability to distinguish the adsorption of H⁺ and OH^- ions from true hydration, i.e., the attachment of water molecules to the protein [23]. Nevertheless, the most serious limitation of this method is that substances added to the protein solution sometimes bind to the protein themselves, resulting in an increase in solubility rather than the expected decrease [24]. Despite these shortcomings, the described method has yielded A number of valuable results. In comparative experiments conducted at various pH values and temperatures, it was found that the non-solvent volume is almost independent of pH [25]. These data indicate that the high viscosity of alkaline protein solutions cannot be attributed to increased hydration, as was previously believed. Another important finding from these studies was the establishment that the non-solvent volume decreases only slightly upon Protein Denaturation and thermal coagulation [26]. It turned out that coagulated proteins retain The ability to bind water to almost the same extent as native dissolved proteins.

Since the hydration phenomenon is accompanied by a decrease in volume, the degree of hydration can be assessed by determining the density of dry proteins and protein solutions using one of the methods described at the beginning of this chapter. The volume contraction accompanying protein hydration can reach 5–8 ml per 100 g of dry protein [27].

In one such experiment, it was found that the density of dry egg albumin suspended in benzene is 1.2715, whereas its apparent density in water is 1.377. This increase in density corresponds to a decrease in specific volume from 0.786 ml per 1 g of dry protein to an apparent volume of 0.726 ml per 1 g of dry protein [28].

The density of most dry proteins is close to 1.27, and the apparent density of proteins in solution is close to 1.34. Plotting the dependence of solution density on the concentration of dissolved protein yields a straight line. The value I — (d_H—d_A)/C_б denotes the density increment; d_н is the density of the undialyzed protein solution, d_Д is the density of the dialyzed protein solution, and c_б is the protein concentration in grams per 1 ml. The density increment is almost independent of either protein concentration or pH, but it depends on the salt concentration in the solution [1]. For carboxyhemoglobin, the density increment I is 0.251 in water and 0.21 in a 1% sodium chloride solution.

J. Adair and M. Adair [1, 29] determined The amount of water bound by protein in solution. These authors measured the density of protein solutions in equilibrium with their respective dialysates. If the protein density increments in solutions containing different amounts of buffer are designated as I₁ and I₂, and the protein densities in the dialysates as d₁ and d₂, then the non-solvent volume is given by V_нр = (І₁— І₂)/(d₂ — d₁). The non-solvent volume comprises the volume occupied by the bound water and the apparent specific volume of the protein, i.e., the volume occupied by the dissolved hydrated protein particles in 1 ml of solution. The amount of bound water can be determined by subtracting the apparent specific volume from the non-solvent volume.

Dissolved serum albumin binds approximately 40 g of water per 100 g of dry protein [1]. This figure is close to the maximum amount of water that 1 g of dry protein can take up (see Fig. 24). Similar results were obtained with other proteins [30]. The amount of bound water ranges from 20 to 50 g per 100 g of protein. A surprisingly low water content (15%) was found in tobacco mosaic virus.

The data presented above show that during the hydration of protein crystals and dissolved proteins, the greatest volume reduction is observed in the region of low water vapor pressure (see Fig. 24). The apparent specific volume of proteins in protein crystals is unlikely to differ from that of proteins in solution. However, both of these values are significantly lower than the specific volume of dry proteins.