HETEROGENEOUS EQUILIBRIUM OF PROTEIN SOLUTIONS II. THE INTERACTION OF CALCIUM CHLORIDE AND OTHER SALTS WITH PROTEINS, AS DETERMINED BY A NEW TYPE OF CALCIUM AMALGAM ELECTRODE BY NORMAN R. JOSEPH (From the Department of Physical Chemistry, Harvard Medical School, Boston) (Received for publication, July 30, 1938) The potentiometric method of studying the interaction of pro- teins and neutral salts has certain important advantages over the two methods that have been principally employed, the solubility method and the study of membrane equilibrium. The solubility method permits the independent variation of the concentration of only one of the components, so that the equilibrium can be studied only at the concentration of a saturated solution of the other. In the method of membrane equilibrium, the concentration of both components can be varied independently, but the equilibrium is attained very sIowly, and except in special cases, as Adair has shown (l), cannot be expressed simply in terms of the activity coefficients of the components. In principle, the potentiometric method is capable of giving directly salt activity as a function of protein concentration and salt concentration, from which data the effect of salt on protein activity and on the membrane equilibrium can be calculated. Studies of this kind have already been reported for the interac- tion of amino acids and salts (9) and for the interaction of protein and salts (10) by means of metallic amalgam electrodes and cells without liquid junction. In the former study it was found that amalgams of the very reactive alkali and alkaline earth metals are not sufficiently stable when exposed to amino acids to yield reliable potentials except under special conditions. They are also un- stable in the presence of ammonium salts and many substances 389 by guest on February 26, 2019 http://www.jbc.org/ Downloaded from
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HETEROGENEOUS EQUILIBRIUM OF PROTEIN SOLUTIONS
II. THE INTERACTION OF CALCIUM CHLORIDE AND OTHER SALTS WITH PROTEINS, AS DETERMINED BY A NEW
TYPE OF CALCIUM AMALGAM ELECTRODE
BY NORMAN R. JOSEPH (From the Department of Physical Chemistry, Harvard Medical School,
Boston)
(Received for publication, July 30, 1938)
The potentiometric method of studying the interaction of pro- teins and neutral salts has certain important advantages over the two methods that have been principally employed, the solubility method and the study of membrane equilibrium. The solubility method permits the independent variation of the concentration of only one of the components, so that the equilibrium can be studied only at the concentration of a saturated solution of the other. In the method of membrane equilibrium, the concentration of both components can be varied independently, but the equilibrium is attained very sIowly, and except in special cases, as Adair has shown (l), cannot be expressed simply in terms of the activity coefficients of the components. In principle, the potentiometric method is capable of giving directly salt activity as a function of protein concentration and salt concentration, from which data the effect of salt on protein activity and on the membrane equilibrium can be calculated.
Studies of this kind have already been reported for the interac- tion of amino acids and salts (9) and for the interaction of protein and salts (10) by means of metallic amalgam electrodes and cells without liquid junction. In the former study it was found that amalgams of the very reactive alkali and alkaline earth metals are not sufficiently stable when exposed to amino acids to yield reliable potentials except under special conditions. They are also un- stable in the presence of ammonium salts and many substances
containing substituted ammonium groups. Attempts to measure calcium ion activity in the presence of protein by means of the flowing calcium amalgam electrode have met with similar difficul- ties (7). Accordingly, in studying the interaction of proteins with ions of the alkali and alkaline earth groups, it is necessary to find a means of protecting the amalgam from protein or other disturbing substances.
In the present paper such a method and its application to several protein systems will be described. In this method the amalgam is protected from protein by a membrane that is permeable only to electrolyte and solvent.
Cells are of the type HgMe ) MeCI, 1 AgCl 1 Ag, the junction be- tween amalgam and solution being made through cellophane. The cell reaction is Me + zAgC1 = MeCl, + xAg (z is the valence of the cation Me).
Assuming the cell reaction to be isothermal and reversible, the electromotive force is given by the relation
where a is the activity of the salt, E” is the standard potential of the cell, v is the number of gm. ions formed by the dissociation of 1 gm. molecule of salt, N is the number of equivalents per mole of salt, and R, T, and F are respectively the gas constant, the ab- solute temperature, and the Faraday constant.
Method
The cell employed is illustrated in Fig. 1. It is an H-shaped tube, in one arm of which a silver-silver chloride electrode is suspended, while in the other the amalgam electrode can be sup- ported. The latter is prepared by sealing platinum wire into glass tubing, and sealing the latter into a calcium chloride drying tube, so that the tip of the platinum comes to within about 0.5 cm. from the mouth of the tube.
Approximately 5 cc. of amalgam are introduced into the tube, the mouth of which is then closed by a cellophane membrane. A square piece of cellophane is moistened and blotted, then stretched tightly over the mouth of the tube, and fixed to the tube by means of collodion. When the tube is inverted and suspended in the
electrode vessel, as illustrated in Fig. 1, the amalgam makes con- tact with the platinum wire and with the solution through the membrane. To renew the surface of the amalgam, it is only neces- sary to remove the tube, invert it so that the amalgam flows into the bulb, and shake the amalgam.
Silver-silver chloride electrodes were prepared by the method of Noyes and Ellis (17), and were frequently checked against each other.
FIG. 1. Cell for determining the activity of salt in the presence of protein. A, amalgam electrode; B, silver-silver chloride electrode; C, cellophane membrane; D, platinum contact.
The amalgams were prepared by electrolysis of the correspond- ing chlorides, redistilled mercury being employed as cathode, in the manner described by Lucasse (13). Care was taken to min- imize exposure of the amalgam to air.
In order to check the method, the activity coefficient of calcium chloride in water has been determined, and compared with the results of Lucasse (13) and Scatchard and Tefft (20), who have employed the procedure of MacInnes and Beattie (14) in which flowing amalgam makes direct contact with the solution,
Our results are given in Table I, at various values of m, the
salt molality. (&.ol-E,) is the difference of E.M.F. obtained when the potential given by a solution of molality m is referred to that of a 0.01 molal solution. Lucasse, in calculating the activity coefficient of calcium chloride, has taken the value 0.716 for the activity coefficient of the 0.01 molal salt, an estimate which is based on the freezing point results of Lewis and Linhart (12). Our values for the activity coefficient are based on the same refer- ence value. As Table I shows, the results of the two methods are in good agreement to 1.0 molal calcium chloride. In pure salt solutions equilibrium across the membrane is usually established within 5 or 10 minutes after immersion of the amalgam electrode.
TABLE I
Activity Coeficient of Calcium Chloride, Obtained from Electromotive Force of Cell,
m
mole per kg. Hz0
0.01 0.02 0.05 0.10
0.20 0.50 1.00
Eo.01 - E
volt 0.0000 0.0230 0.0450 0.0764 0.0994 0.1360 0.1770
* Observed with flowing amalgam. The results of Fosbinder (7) are in good agreement with both sets of observations up to 1.0 M Ca&.
t Reference value based on freezing point data (12).
The observed potentials often have a tendency to drift. This usually indicates a certain amount of oxidation at the surface of the amalgam. The potentials are restored to the initial value when the amalgam is shaken and a fresh surface restored. By such means it is usually possible to obtain reproducible potentials that vary only within 0.5 millivolt. Each E.M.F. that is given represents the mean of four or more successive approximately constant readings taken over a period of at least 15 minutes. Most of them were carried out in duplicate. The agreement of the results obtained by this method with those of the flowing amalgam technique indicates that there is no significant potential introduced into the cell by the membrane itself.
Another check of the method in a protein system may be ob- tained by studying the activity of zinc chloride in the presence of isoelectric gelatin, and comparing the results with those obtained with flowing amalgams (10). This comparison is presented in Table II. It also indicates good agreement between the two methods, and is evidence that no error is introduced into the ob- served E. M. F. by the membrane itself.
In the case of membrane equilibrium in isoelectric protein solu- tions it is well known that the membrane potential may amount to many millivolts (15). It is therefore important to consider the
TABLE II Interaction of Zinc Chloride and Gelatin Determined by Two Types of Zinc
Amalgam Electrodes
ma Protein E,-E,,’ Et
mole salt per kg. Hz0 gm per kg. Hz0 volt 0.01 50 0.0064 0.01 75 0.0098 0.04 50 0.0030 0.04 75 0.0048 0.10 50 0.0019 0.10 75 0.0028 0.25 50 0.0012 0.25 75 0.0017
udt
0.0070 0.0100 0.0034 0.0050 0.0016 0.0024
-
* Observed with the electrode making contact through the membrane. t Observed or estimated from data with flowing zinc amalgam electrode
at 37” (10).
relation between the usual type of membrane potential and the potential measured in these experiments.
The accompanying diagram illustrates the type of equilibrium with which we are dealing.
It is a condition of equihbrium of this system that the chemica1 potential of the salt FMextA) in phase A must be equal to FM~X~C), the chemical potential of salt within the membrane C. Let US consider the change of state resulting from the passage of 1 equiv- alent through the cell, assuming it to function isothermally and reversibly. At the junction of membrane and the amalgam elec- trode B, 1 equivalent of the cation Me is formed. The correspond- ing single electrode potential is
RT EMe(C) = &e - - NF In aMe (1-a)
where o&(C) is the activity of cations within the membrane, and EL, is the standard single electrode potential of the amalgam.
At the Ag,AgX electrode, 1 equivalent of the anion X is formed and the electrode potential is
RT Ex(A) = E% - zln ax(A) 0-b)
where ox(A) is the activity of anions in the solution and E& is the standard potential of the electrode.
To fuIfi1 the condition of electrica neutrality in phases A and C, it is necessary that 2 equivalents of the cation migrate from C to A, and that (1 - z) equivalents of the anion migrate from A to C. The net process is the isothermal and reversible formation of zr equivalents of MeX in phase A and (1 - z) equivalents within the membrane. It is evident that x is a function of the relative quan- tities of electrolyte in A and C, and that as the ratio of the volume of A to that of C approaches infinity x approaches unity. This condition can be assumed to be met in the system we are consider- ing, for C is a thin membrane and contains a very small mass of electrolyte in comparison with the relatively large mass in A. Thus for every equivalent passing through the cell, 1 equivalent of the cation Me migrates from C to A, while an infinitely small quantity of the anion X is transferred to C.
At the membrane there must be a potential given by the rela- tion
This is the potential determined in studies of membrane equilib- rium. Adding the three potentials given by Equations l-a, l-b, and l-c, one finds
E =I Eo - g In WfeX(A)
where EQ = E& + Ek. This is identical with Equation 1. The E.M.F. of the cell is
determined therefore by the activity of salt in A and by the stand- ard electrode potentials, and is not affected by the membrane it- self as long as the ions within the membrane are in equilibrium with those in the solution. Under these conditions the cell reac- tion can be regarded simply as the formation of salt in the solution from the metal and the silver salt, the free energy change of the process being independent of t.he steps.
Application to Protein Solutions
Determinations of salt activity have been obtained in solutions of the following proteins: horse carboxyhemoglobin, horse serum albumin, pseudoglobulin, and gelatin. The serum albumin and pseudoglobulin were electrodialyzed preparations for which I am indebted to Dr. John D. Ferry. The former was Preparation IIIDz described by Ferry and Oncley (6). An approximately 5 per cent solution had a specific conductivity of 52 X lo+ reciprocal ohm. The dielectric increment per gm. of this fraction per liter of solution has been found to be 0.29. A 2 per cent solution of the pseudoglobulin had a specific conductivity of 9 X lo+ recipro- cal ohm. The dielectric increment per gm. of this fraction per liter of solution is 0.9,l at infinite dilution of protein. This is somewhat lower than the value at infinite dilution obtained for the fractions described by Ferry and Oncley, and indicates the presence in this preparation of small amounts of other globulins.
Carboxyhemoglobin was prepared from horse red blood cells according to the directions of Stadie and Sunderman (21), with a different method of electrodialysis which will be described else- where. The specific conductivity of an approximately 1.5 per cent solution was 45 X lo-+ reciprocal ohm.
Gelatin was prepared according to the directions of Northrop
(10) that Equation 3 is correlated by thermodynamics with the equation
0.2 0.2 0.4 0.4 0.6 0.6 Cl8 Cl8
(4)
FIG. 2. The influence of calcium chloride on the activity coefficients of various proteins at 25”, as estimated from the effects of the proteins on the activity of the salt.
where yz and yzo are, respectively, protein activity coefficients in the presence of salt and in pure water. The solubilities of proteins in the presence of salts have been described by equations analogous to Equation 4 (3, 8, 19).
According to Ferry, Cohn, and Newman (5), this is an empiri- cally valid method of characterizing solubility data. Although their results on the solubility of hemoglobin in ethanol-water mixtures can be characterized by some form of Equation 4, they consider it of greater theoretical significance to express their re- sults in the form given by the Kirkwood theory (11) for the elec- trostatic interaction of ions and complex dipoles. This, as they have shown, leads to a linear relation at very low concentrations of salt, but approximates a square root formulation at higher con- centrations. Their treatment involves also the consideration of non-electrostatic forces. Accordingly in Table VII, -log y.Jvz” is given as a function of p, the ionic strength, which for the systems under consideration is approximately equal to I’/2, the concentra- tion unit by which Ferry, Cohn, and Newman describe their re- sults. These results are given graphically in Fig. 2, which illus- trates the effect of calcium chloride on the activity coefficients of the proteins.
DISCUSSION
In all the systems studied, the effect of the salt is to reduce the activity coefficient of the protein, according to the empirical Equa- tion 4. The magnitude of this effect varies widely for different proteins, as solubility studies have also indicated, increasing in the order, carboxyhemoglobin, serum albumin, gelatin, pseudo- globulin. The calculated results for carboxyhemoglobin indicate effects of the same order of magnitude as those obtained by solu- bility studies on this protein (5,8,22). They are, however, based on E.M.F. of about 1 millivolt, and are not of great significance. The small E.M.F.'S obtained with t,his protein are related to its low solubility, and correspond to its negligible effect on the osmotic coefficient of sodium chloride as determined by the freezing point method (21). The results for t,he other proteins are more signifi- cant, since for all of them potentials of at least 3 or 4 millivolts could be obtained.
That there is a correspondence between the activity coefficients and other electrostatic properties of the proteins is indicated by consideration of the values of the molal dielectric increments of the proteins. The molal dielectric increment 6 is approximately 22,000 for carboxyhemoglobin (18), 20,000 for the most polar
serum albumin fraction studied by Ferry and Oncley (6), and 135,000 for the pseudoglobulin fraction employed in the present studies.l The value of 6 for gelatin is of the order of 90,000 (4). Thus, there appears to be a rough parallel between the molal dielectric increments of the proteins and their interaction with neutral salts. The relation between these two effects has been thoroughly discussed by Cohn (2) for the case of amino acids and peptides. The results of the present study appear to indicate a general parallelism between 6 and the salt effects, even for such highly complex molecules as the proteins.
The author wishes to express his appreciation to Professor Edwin J. Cohn for his interest in this work, and to Professor George Scat- chard for his criticisms of the manuscript.
SUMMARY
1. A new type of amalgam electrode for studying the interaction of proteins and neutral salts is described. Decomposition of the amalgam by protein is prevented by a cellophane membrane, which is equilibrated with the solution.
2. It is shown theoretically that such a membrane, provided it be in equilibrium with the solution, introduces no significant po- tential into the cell.
3. The E.M.F.‘S obtained with this electrode are shown to agree with those obtained with flowing amalgam electrodes.
4. The effects of several proteins on the activity of calcium chloride and other salts have been determined.
5. From the potentiometric dat.a, the effects of calcium chlor- ide and the other salts on the activity coefficients have been deter- mined. These effects are comparable with the results of solubility studies.
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