Chemistry and Biology of Proteins - F. Haurowitz 1953

Electrochemistry of Proteins
Binding of Proteins to Other Ions

As noted in the previous section (see also p. 221), Proteins combine not only with hydrogen ions but also with other ions present in solution, and this phenomenon affects the electrometric titration and the Determination of the isoelectric point of proteins. Protein-ion binding is driven by the electrostatic forces of ionized groups: anions bind to the positively charged groups of the protein, whereas cations are attracted by the negatively charged groups The binding of extraneous ions by protein molecules is of profound biological significance, as a substantial fraction of the inorganic ions present in tissue fluids is bound in precisely this manner.

The presence of ion-Structure/178.html">Protein Complexes in tissue fluids can be demonstrated by dialysis against a protein-free solution containing the same ion at an identical concentration [39]. If the ion in the tissue fluid exists in an unbound state, no change in its concentration will be observed. Conversely, if a portion of the ions is bound to the protein, ions will migrate from the dialysate into the tissue fluid until equilibrium between free ions is established on both sides of the membrane.

The binding of inorganic ions to protein molecules is also revealed in Electrophoresis experiments. In such experiments, the migration of cations (such as Ca⁺⁺) toward the anode, or anions toward the cathode, is frequently observed, indicating that the ion is bound to the protein molecule and that the resulting complex moves as a single entity.

The binding of various ions by proteins depends primarily on their valence. Monovalent alkali Metal Ions and chloride ions are bound only to a negligible degree, and the bulk of them remains in a free state in tissue fluids. In contrast, calcium, magnesium, and phosphate ions in tissue fluids are largely bound to proteins.

The fact that proteins are precipitated by heavy metal salts also indicates that the ions of these metals form numerous stable bonds with proteins. The binding of inorganic and organic ions by proteins occurs not only in physiological fluids but also in vitro when Buffer solutions are used. To prevent the association of ions with proteins, it is necessary to use buffer solutions composed of salts with monovalent ions, such as alkali metal salts of acetic, diethylbarbituric, or monobasic phthalic acids.

Among the ions that form compounds with proteins, calcium has been studied with particular thoroughness. Approximately 40% of the calcium in Muscle serum [40] and roughly one-third of the calcium in Blood serum are bound to protein molecules [41]. One gram of Serum proteins is capable of binding 0.062 mM of calcium [42]. The combination of proteins with Ca⁺⁺ proceeds According to the law of mass action [43]. In milk, a significant portion of the calcium is bound to the phosphate groups of casein [44]. From a physicochemical standpoint, magnesium behaves similarly to calcium, and substantial quantities of magnesium ions are bound by protein molecules in both muscle serum and plasma serum [40, 41].

In the physicochemical equilibrium of blood serum, the binding of phosphate to protein molecules is of minor importance, given the very low phosphate content in serum.

The combination of carbon dioxide with proteins is of major importance. Siegfried [46] demonstrated that Amino Acids combine with carbonates to form carbamino acids

Class="center">

This reaction occurs exclusively in alkaline solutions. It is believed that a fraction of the carbon dioxide present in blood combines with the protein component of Hemoglobin in an analogous manner, forming carbaminohemoglobin [47–52]. This hypothesis is supported by the observation that blood binds carbon dioxide in two phases; the second, slower phase corresponds to the well-known slow conversion of carbon dioxide into bicarbonate. This phase is preceded by a rapid uptake of carbon dioxide, which is attributed to The formation of a carbamino compound. Indeed, it has been established that a portion of the carbon dioxide cannot be precipitated by barium hydroxide.

It should be borne in mind, however, that typical carbamate formation takes place only in strongly alkaline solutions, where the ammonium group N⁺H₃ is converted into the amino group NH₂; therefore, it remains unclear whether carbamino compounds can be formed in appreciable amounts under physiological conditions. It must also be taken into account that bicarbonate ions can react with positively charged protein groups according to the formula

This is observed in electrodialyzed, salt-free protein solutions [20, 53]. The combination of carbon dioxide with hemoglobin plays a crucial role in the physicochemical equilibrium of blood, since the presence of carbon dioxide reduces the oxygen affinity of hemoglobin—a phenomenon well established in physiology. The Effect of carbon dioxide on hemoglobin's oxygen affinity is due to the fact that carbon dioxide binds to basic amino groups or imidazole groups located in the immediate vicinity of the iron atom to which oxygen attaches [52, 54].

Electrometric titration and electrical conductivity measurements of protein solutions in the presence of alkali metal chlorides have shown that small amounts of these monovalent ions are also bound to proteins [27, 55, 56]. In blood serum, however, this quantity is so small that it can be detected neither by compensation dialysis [39] nor by electrophoretic transference number measurements [57]. Conversely, data from electrometric titration and conductivity determinations have demonstrated that approximately 10 Functional groups of the serum albumin molecule combine fairly strongly with chloride or thiocyanate anions, and that, in addition, a significant number of these anions are bound to the protein via weaker linkages [27]. The maximum number of protein-bound ions correlates well with the number of oppositely charged groups present in the protein molecule. For instance, it has been established that egg albumin combines with metaphosphoric acid to yield a crystalline compound containing approximately one molecule of metaphosphoric acid for each positively charged protein group [58].

Among protein compounds with heavy metals, naturally occurring copper-protein complexes deserve mention, such as hemocuprein from bovine blood erythrocytes [59], hepatocuprein from the Liver [59], hemocyanins from the blood of certain invertebrate species (see Chapter XI), and copper-containing oxidases [60]. Copper is present not only in soluble liver proteins but also in the insoluble residue obtained after extraction with dilute ammonia [61]. Apparently, in all these compounds, copper ions are bound to negatively charged protein groups. Proteins combine with copper in a similar fashion in vitro as well. The binding of copper by serum albumin is accompanied by a release of Free energy; ∆F decreases from —5,179 cal per eq upon the attachment of the first copper ion to —1,271 cal upon the binding of the sixteenth copper ion. The concurrent increase in Entropy indicates that Water of Hydration molecules are displaced by copper ions and their bonds with the protein are disrupted [62].