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(1) CARR,C. W.. AND SOLLNER, K . : J. Gen. Physiol. 26, 119 (1944). H . P., AND SOLLNER, K.: J. Gen. Physiol. 26,179 (1945). (2) CARR,C. W., GREQOR, (3) COLLANDER, R.:Kolloidchem. Beihefte 19, 72 (1924);500. Sci. Fennica, Commentationes Biol. 2, 6 (1926). (4)DOLEZALEK, F.,AND KRUGER,F.: Z. Elektrochem. 12.669 (19%). (5) GREGOR, H.P.: Ph. D.Thesis, University of Minnesota, 1945. (6) GREOOR,H . P., AND SOLLNER, K.: J. Phys. Chem. 60, 53 (1946). (7) GREGOR,H . P., AND SOLLNER, K.: J. Phys. Chem. 60, 88 (1946). (8) GROLLMAN, A., AND SOLLNER, K.: Trans. Electrochem. SOC.61, 487 (1932). (9) H ~ B E RRUDOLF: , Physical Chemistry of Cells and Tissues. The Blakiston Company, Philadelphia (1945). (10) MICHAELIS,L.:Bull. Natl. Research Council (U. 5.) No. 69, 119 (1929). (11) SOLLNER, K.:Z.Elektrochem. 98, 36,234 (1930);Kolloid-Z. 62,31 (1933). (12)SOLLNER, K.: Biochem. Z. 244, 370 (1932). (13) SOLLNER, K., AND GREQOR, H. P.: J . Am. Chem. 500. 67, 346 (1945). (14) SOLLNER, K., AND GREGOR, H. P.: J. Phys. Chem. 60,470 (1946). (15) SOLLNER, K., AND GREGOR,H . P.: J. Phys. Chem. 61, 299 (1947). (16) SOLLNER, K., AND GREGOR,H . P.: J. Phys. & Colloid Chem. 64 (March, 1950). (17)SOLLNER, K., AND GREGOR, H . P.: In preparation. (18) SOLLNER, K . , AND GROLLMAN, A.: Z. Elektrochem. 88, 274 (1932). L.: J. Gen. Physiol. 12, 55 (1928). (19) WEECH,A. A,, AND MICHAELIS,
T H E DETERMINATION OF SODIUM-ION AND CHLORIDE-ION ACTIVITIES IN PROTEIN SOLUTIONS BY MEANS OF PERMSELECTIVE MEMBRANES’ CHARLES W. CARR
Department of Physiological Chemistry, The Medical School, University of Minnesota, Minneapolis 14, Minnesota AND
LEO TOPOL Division of Physical Chemistry, School of Chemistry, Institute of Technology, University of Minnesota, Minneapolis 14, Minnesota Received August 8.9, 10.43
I The study of the interaction of various ions with proteins in solution has been of considerable interest for many years. It is now well known that the activity of many ionic substances is markedly influenced by the presence of proteins, the most familiar case being that of the binding of acids and bases. Through the use of various experimental techniques, it has also been shown that the activity of 1 Presented at the Twenty-third Kational Colloid Symposium, which was held under the auspices of the Division of Colloid Chemistry of the American Chemical Society at Minneapolis, Minnesota, June 6-8, 1949.
ION ACTIVITIES I N PROTEIK SOLUTIONS
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many organic ions, heavy metal cations, and alkaline earth cations is decreased considerably in protein solutions, owing t o combination with the proteins. Recently it has been found that many of the common anions, including chloride, are bound to a certain extent to proteins (10, 13, 24, 27, 28). There are some indications that the alkali metal cations may also interact with some proteins, but this problem has not been completely elucidated (4, 8, 11, 22). It is the intention of the present paper to show how membrane electrodes might be applied t o the study of the interaction of the alkali metal cations and halogen anions with proteins. With the use of such electrodes it is possible to measure directly the activity of certain ions. In experiments of this nature any decrease in activity of a given species of ions in a protein solution below that which it shows in a solution of the same ionic concentration without protein can be taken as an indication of some type of interaction between the ions and the proteins. Specific electrodes for the determination of the activities of sodium and chloride ions in protein solutions have occasionally been used in the past. Several workers have used the sodium amalgam electrode t o study the sodium-ion activitv in blood serum (19,20,23).Although the presence of proteins increases considerably the error involved in the use of this electrode, it has been the general conclusion that the sodium-ion activity in serum is unaffected by the presence of the proteins. Kirk and Schmidt (9) have used this electrode in solutions of sodium caseinate. Their results showed that the casein also had no detectable effect on the activity of sodium ions. Because of the extreme care necessary for their preparation and use and the error introduced by the presence of protein, the amalgam electrodes are limited in their usefulness for such studies. Hitchcock (7) has used the silver-silver chloride electrode to measure the chloride-ion activity in gelatin solutions. He worked a t relatively low pH’s (1-2) equivalents per gram) was and found that a small amount of chloride (2 X bound t o the gelatin. Northrop and Kunitz (21) also used this electrode in solutions of gelatin and found that a t a higher pH (4.7) no detectable chloride was bound in a 1 per cent solution of gelatin. At higher concentrations (6-15 per cent) they found the chloride-ion activity t o decrease slightly below that found in salt solution without protein. Membrane electrodes for the determination of ion activities other than hydrogen have just recently been developed. Sollner (26) and Gregor (5) have shown that specially prepared permselective collodion membranes may be used for the determination of some of the more common cations, including sodium, potassium, ammonium, and magnesium. Similarly, permselective protamine collodion membranes may be used for the determination of the anions chloride, bromide, nitrate, acetate, and several others. Marshall and his collaborators (14, 15, 16, 17, 18) have used clay membranes successfully for the activity determinations of sodium, potassium, ammonium, magnesium, and calcium ions. Wyllie and Patnode (29) very recently have prepared an artificial membrane by bonding a cation-exchange resin in an inert plastic. Their membranes have been found to give good results for the determination of sodium-ion activities in solutions as concentrated as 4 molal sodium chloride.
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The membranes used in the present work are the collodion and protamine collodion membranes, and the ion activities are determined by the titration procedure of Sollner (26). The proteins used are gelatin and casein, and the electrolyte used is sodium chloride.
I1 The determinations of ion activity are made by carrying out a potentiometric titration. The solution of unknown activity is placed on one side of the membrane and a known volume of water on the other side. A strong solution of known concentration of an electrolyte which has the same ion as the one being determined is added to the water from a buret. After each addition of electrolyte the membrane potential is measured, and the addition of electrolyte is continued until the potential changes in sign. At the point of zero potential the activity of the ion under consideration is equal to that of the same ion in the outside solution. From the volume and concentration of strong electrolyte added to a given volume of water, the concentration of this electrolyte a t the zero potential is calculated, and from this concentration the activity of the ion under investigation is estimated. The exact procedure for carrying out the titrations was similar to that described previously (2). The membranes which were used were prepared as described by Sollner and coworkers (1, 3, 6). They are bag-shaped, being formed over 25 x 100 mm. test tubes, and hold about 30 ml. of solution. For the determination of cation activity the oxidized collodion membranes are used. These membranes are negatively charged with respect to the solutions in which they are immersed. For the determination of anion activity the positively charged protamine collodion membranes are used. A known volume of water is added to a beaker of such size that the beaker is about one-half to two-thirds filled. Next, the membrane is filled with the solution t o be investigated. About two-thirds of its length is immersed in the beaker containing the water, and the membrane is then clamped in position. For a clamp a wooden test tube holder held t o a ringstand with a right-angle clamp is convenient. The temperature is measured and titration is started. The electrolyte solution used as titrant should be a t least ten times as concentrated as the solution being titrated t o avoid the addition of excessively large volumes which would be needed to reach the end point. After each addition the E.M.F. of the system is determined: outside inside HglHg,C12(s) KCI(satd.) solution membrane solution KCl(satd.) Hg?ClZ(s) IHg l
C
L
1
i c z l
The saturated calomel electrodes are connected with the two solutions by means of specially constructed agar bridges saturated with potassium chloride. These bridges are made with 3-mm. glass tubing, and the tips which make contact with the solutions are about 1 mm. in diameter, The small contact area is necessary
IOX ACTIVITIES IN PROTEJY SOLUTIOXS
179
to minimize the diffusion of potassium chloride into either solution. To minimize this diffusion still further, the bridges are removed from the two solutions after each measurement of the potential and placed in saturated potassium chloride solution, Just before use they are wiped dry and placed in the solutions being measured. In making the potential measurements, two readings were always taken. After the first reading the agar-potassium chloride bridges were changed around so that the solutions with which they were in contact became reversed. A second reading was then taken, which usually varied from the first by a few tenths of a millivolt. The average of the two readings was taken to be the observed potential. This potential was plotted on the linear axis of semilogarithmic graph paper, and the concentration of the outside solution was plotted on the logarithmic axis. A straight line with nearly the theoretical slope was always obtained. The intersection of this line with the line of zero potential thus gives the concentration in the outside solution which corresponds to the zero potential. This concentration a t which the zero potential is obtained will hereafter be referred to as the end point of the titration. For known solutions in the range of concentration of 0.005 M to 0.2 M , the correct end point can be reproduced with an accuracy of about f l - 2 per cent. Since the protein solutions which we intended to study vary considerably in pH, we have determined the pH range in which the membranes can be used without introducing serious error. Sodium chloride solutions (0.100 N ) were prepared, and the pH of these solutions was varied by addition of small amounts of hydrochloric acid or sodium hydroxide. Although the pH values of these unbuffered solutions were not known very accurately, they were known with sufficient accuracy to determine in which range of pH the membranes would be useful. The solutions were titrated immediately after preparation with the use of a neutral solution of 1 sodium chloride as the titrant. It was found that when 0.1 N sodium chloride was placed in a negative membrane, the end point was reached after the correct amount of titrant was added only when the pH was greater than 4.5. As the pH decreased below 4.5, it required more than the correct amount of I N sodium chloride to reach the end point. The correct end point was also reached when the pH was as high as 10.0. I t was concluded that the negative membranes could be used successfully for the determination of sodium ion over a pH range of 4.5-10.0. When the 0.1 N sodium chloride was placed in a positive membrane, the correct end point was reached a t a pH of 9.5 but not at 10.0. The correct end point was also reached a t a pH as low as 3.0. I t appears that the positive membranes can be used successfully for the determination of chloride ion over a pH range of 3.0-9.5.
The proteins used in these experiments were commercial preparations which were purified further to make sure that the electrolyte content was negligible. The gelatin mas purified by a method outlined by Schmidt (25). It was washed with dilute acetic acid at 5OC., washed with water a t the same temperature, and electrodialyzed. I t was then washed with ethanol and ether and finally dried. A
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2 per cent stock solution of the gelatin and a 0.300 N stock solutioh of sodium chloride were prepared. Known volumes of the two solutions were mixed so that when the final dilution was made, the concentration of gelatin was 1.0 per cent and the concentration of sodium chloride was 0.100 N . Just before the final dilution was made the pH was adjusted to approximately the desired value by the addition of a small amount of either standard hydrochloric acid or standard sodium hydroxide. Thus, except in one instance the final sodium-ion concentration was not exactly equal to the chloride-ion concentration. In all cases the pH of the solution as it was finally prepared was measured with a pH meter. The pH of these solutions was varied from 4.0 to 8.2. A second series of 1.0 per cent gelatin solutions was also prepared in which the final concentration of sodium chloride was 0.0100 N and in which the p H varied from 3.9 to 9.1. The casein was precipitated a t its isoelectric point by the addition of acid t o an alkaline solution of casein. The precipitate was then thoroughly washed and finally extracted with methanol and dried. Solutions were prepared by adding a known amount of standard sodium hydroxide to weighed amounts of casein, and the solutions were then brought t o approximately the desired pH by the addition of standard hydrochloric acid. Distilled water was then added so that the final casein concentration wm always 1.0 per cent. The amount of standard base was chosen so that the final concentration of sodium was 0.100 N in one series and 0.0100 N in the other series. The chloride concentration varied from these figures, depending on the amount of standard acid needed to reach the desired pH. For the solution on the acid side of the isoelectric point, it was necessary t o dissolve the casein in 0.0100 N hydrochloric acid and adjust the pH by the addition of standard sodium hydroxide. In all cases the pH of each solution was measured after the final dilution. The variation in pH was over the range 3.9-9.1.
I11 We turn now to the measurement of the sodium chloride solutions containing 1 per cent gelatin and 1 per cent casein, respectively, the results of these determinations being given in tables 1-4. The first column in each table shows the pH of the solutions as measured with the p H meter. The second column gives the known concentration of the ion being studied that was added to the protein solution. In the third column is the activity of that ion calculated on the basis that it behaves as a completely dissociated ion in a solution of a uni-univalent electrolyte of the concentration given in column 2. To make this calculation, we have used the single ion activity coefficients as given by Lewis and Randall (12). Thus the figures in column 3 of the tables are obtained by multiplying the known concentration given in column 2 by the activity coefficient a t that concentration. In the fourth column is the measured activity of the ion being determined in the protein solutions. These values are obtained by multiplying the end point concentration of sodium chloride in the solution containing no protein by the activity coefficient for the ion in question. The ionic activity coefficients used here are the same as those used for the figures in column 3.
181
IOK ACTIVITIES IEi PROTEIS SOLrTIOEiS
TABLE 1 Determination oj the chloride-ion actii3ilies in 1 p e r cent yelatiu solutions containing known concentrations of sodiuni chloride (1)
(2'
i
I CONCESTR. T I Y OF CELOPIDE 10s ACTIVIIY
~
9.0 7.6 5 9
0.087 0.091
5.2 3.9
0,0074
0,095
0.01cQ
0.069 0.073 0.075
0.070 0.074 0 .074
1.01 1.01 0.99
0.0069 0.0092
0,0068 0.0094
0.99 1.02
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CHARLES W. C I R R AND LEO TOPOL
Finally, in column 5 we have the ratio of the figures in columns 4 and 3, measured ion activity/calculated ion activity; this ratio is called the relative ion activity. If the relative activity is 1.00 i= 0.02, then me can conclude that the presence of the protein in the sodium chloride solution has not significantly changed the ionic activity. A value of less than 1.0 for this ratio indicates some kind of effect on the activity of the ion in question. It should be pointed out here that any error in the single ion activity coefficients which we have used will cause no significant change in the ratios of column 5. These activity coefficients,based on extrathermodynamic assumptions, are used for both values in the ratio, and any error would cancel out. In other words, the results which we obtain are not necessarily the absolute ion activities but are only relative to sodium chloride solutions of known concentration. TABLE 4 Determination o j the sodium-ion activities in 1 per cent casein solirtions containiny known concentrations of s o d i u m chloride (3) CONCENTPATION OF ADDED S O D I M I O N
'AICULATED ACTIVITY OF S O D I M ION
(4) MEASURED ACTIVITY OF S O D I M ION
(5) LELATIVE SODIUM-ION ACllnTY: YEASUPED LCTIYITY/CAICQLATED ACTIVITY
9.0 7.6 5.9
0.098 0.088 0.100
0.078 0.071 0.080
0.076 0.069 0.080
0.97 0.97 1.00
8.7 8.0 7.0 5.2
0.0100 0.0100 0.0100 0.0100
0.0092 0.0092 0.0092 0.0092
0.0069 0,0072 0. 0082 0.0093
0.75 0.78 0.89 1.01
IV The data of tables 1 and 2 show quite clearly that for every solution tested the activities of both chloride ion and sodium ion are unchanged by the presence of the gelatin. Even in the 0.0100 N solutions, the measured ion activities are the same as the calculated ion activities over a pH range of 4-9. The results in table 3 show also that there is no measurable effect of casein on the activity of chloride ions in solution. Over a pH range of 3.9-9.0 and in 0.1 N and 0.01 N solutions the activity of the chloride ion is the same as in known sodium chloride solutions containing no casein. With regard to the sodium ion in casein solutions, a different situation appears t o exist. In solutions 0.1 N in sodium, at a pH above 7, there may be some interaction. The decrease in the values of the relative activities is just on the border line of being significantly lower than 1.0. In the more dilute solutions, however, it is quite evident that some kind of an effect exists at a pH of 7 and above. At a pH of 5.2 the relative activity is 1.01. As the pH increases the ratio becomes smaller and smaller. At pH 7 the activity of sodium ion in the outside solution
IOX ACTIVITIES IN PROTEIN SOLUTIONS
183
a t the end point of the titration is 11 per cent lower than the calculated activity of sodium ion in the casein solution. At pH 8.7 this difference has increased to 25 per cent. Further experiments to confirm this effect for casein solutions have indicated that this decrease in sodium-ion activity is directly proportional to the amount of casein present. For example, a t pH 7.5 and 0.02 N sodium concentration, the difference between the measured sodium-ion activity and the calculated sodiumion activity in a 1 per cent casein solution is equivalent to 0.18 milliequivalent of sodium per gram of casein. In a 2 per cent casein solution of the same pH and sodium concentration this figure is 0.21 milliequivalent per gram, and in a 4 per cent casein solution it is 0.20 milliequivalent per gram. These three values are the same within the limits of error of the experiments. Kirk and Schmidt (9) in their work measured the sodium-ion activity in casein solutions only at a pH of about 5.5. Their fesults also showed that in this pH range the casein has no detectable effect on the activity of sodium ions. They did not, however, make any measurements at higher pH’s. From our results it appears that in casein solutions which are slightly alkaline, the activity of sodium ions is appreciably depressed. However, further experiments will be necessary to confirm this point. For all the other cases studied the presence of either gelatin or casein does not affect the activity of either the sodium or the chloride ions. These experiments indicate that the method offers a relatively simple technique for studying the problem of the interaction of proteins with small ions. For example, it would be of interest to confirm the binding of chloride and other small ions to serum albumin (24) by the use of this method. Another problem which can be attacked is the determination of the effect of the muscle proteins on the activity of potassium ion (4, 11, 22). Similarly, one could study the activity of ions in solutions of purified enzymes, especially those enzymes which are activated by the presence of such ions as magnesium, calcium, or chloride. Therefore we hope to continue this work along these lines. SUMMARY
Specially prepared collodion membranes have been used as electrodes for the determination of sodium-ion activity in protein solutions. Similarly, protamine collodion membranes have been used for the determination of chloride-ion activity. In 1 per cent solutions of gelatin containing known concentrations of sodium chloride, the activity of both the sodium and the chloride ions was found to be the same as in solutions of sodium chloride of the same concentration containing no protein. The concentrations of sodium chloride used were 0.1 N and 0.01 N ; the pH of the solutions was varied from 3.9 to 9.1. In 1 per cent solutions of casein containing known concentrations of sodium chloride, the activity of the chloride ions was found to be unaffected by the presence of the protein. The variation in salt concentration and pH was the same as for the gelatin experiments. The sodium-ion activity was the same as in
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protein-free sodium chloride solutions a t pH 5.2. At pH 7 and in 0.01 N sodium chloride solutions, however, the sodium-ion activity was found to be 11 per cent lower and at pH 8.7 was 25 per cent lower than in sodium chloride containing no casein. The results indicate that the method will be applicable to the determination of the activity of many small ions in the presence of any water-soluble protein. The authors wish to thank Dr. Karl Sollner for his helpful suggestions and criticisms in the preparation of the manuscript. REFERESCES CARR,C. W., GREGOR, H. P., AND SOLLTER,K . : J. Gen. Physiol. 28, 179 (1945). CARR,C. W., JOANSON, W. F., AKD KOLTHOFF, I. 31.:J. Phys. & Colloid Chem. 61, 636 (1947). CARR,C. W., AND SOLLNER, K . : J. Gen. Physiol. 28, 119 (1944). FENN, W. 0 . : Physiol. Rev. 16, 450 (1936). GREGOR, H . P . : Ph.D. Thesis, University of Minnesota, 1945. GREGOR, H. P., AND SOLLNER, K.: J. Phys. Chem. 60,53,88 (1946). HITCHCOCK, D. I . : J. Gen. Physiol. 12, 495 (1928). INGRAHAM, R. c . , LOMBaRD, c . , AND VISSCHER, M. B.: J. Gen. Physiol. 16, 637 (193233). KIRX,P. L., AND SCHMIDT, C. L. A , : J. Biol. Chem. 76,115 (1928). KLOTZ, I. &I., AND URQUHART, J. M.:J. Phys. & Colloid Chem. 63, 100 (1949). KOhfETIANI, P. A . : Biokhimiya 13, 137 (1948). LEWIS,G. K . , AND RASDALL, M.: Thermodynamics, p. 382. McGraw-Hill Book Cornpany, Inc., Xew York (1923). LONGSWORTH, L. G., AND JACOBSEN, C. F.: J. Phys. & Colloid Chem. 63,126 (1949). MARSHALL, C. E., AND AYERS,A. D., J. Am. Chem. SOC.70,1297 (1948). MARSHALL, C. E., AND BERGMAN, W. E . : J. Am. Chem. SOC.63, 1911 (1941). MARSHALL, C. E., AND BERGMAN, W. E.: J. Phys. Chem. 48,325 (1942). MARSHALL, C. E., AXD EIME,L. 0.:J. Am. Chem. SOC.70, 1302 (1948). MARSHALL, C. E., AND KRINBILL, C. A , : J. Am. Chem. SOC.64,1814 (1942). MICHAELIS, L., AND KAWAI,S.: Biochem. 2. 163, 1 (1925). KEUHAIJSEN,B. S., AND MARSHALL, E. K., JR.: J. Biol. Chem. 63,365 (1922). NORTHROP, J. H., AND KWNITZ, M.: J. Gen. Physiol. 7, 25 (1924-25). PETERS, J. P.: Physiol. Rev. 24, 491 (1944). RINGER,W. 2.: Z. physiol. Chem. 130, 270 (1923). SCATCHARD, G., AND BLACK,E. S.: J. Phys. & Colloid Chem. 63, 88 (1949). ScHnrIDT, C. L. A , : Chemistry of the Amino Acids and Proteins, p. 174. Charles c. Thomas, Baltimore (1944). SOLLNER, K.. J. Am. Chem. SOC.66, 2260 (1943). STEINHARDT, J.: Ann. N. Y. Acad. Sci. 41,287 (1941). VELICK,S. F.: J. Phys. & Colloid Chem. 63, 135 (1949). WYLLIE,M. J. R., AND PATNODE, H. W.: J. Phys. & Colloid Chern. 64, 204 (19%).
