THE APPARENT DISSOCIATION CONSTANTS OF ARGI- NINE AND OF LYSINE AND THE APPARENT HEATS OF IONIZATION OF CERTAIN AMINO ACIDS.*

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1 THE APPARENT DISSOCIATION CONSTANTS OF ARGI- NINE AND OF LYSINE AND THE APPARENT HEATS OF IONIZATION OF CERTAIN AMINO ACIDS.* BY CARL L. A. SCHMIDT, PAUL L. KIRK, AND W. K. APPLEMAN. (From the Division of Biochemistry, University of California Medical School, Berkeley.) (Received for publication, May 19, 1930.) In this paper the apparent dissociation constants of arginine and of lysine at 25 and the apparent dissociation constants of arginine, lysine, histidine, aspartic acid, and glutamic acid at 0 are reported. We have made use of these data together with previously published data for the apparent dissociation constants of histidine, aspartic acid, and glutamic acid for the purpose of calculating the apparent heats of ionization of these amino acids. The technique employed for obtaining the titration curves was the same as that which has been described by Kirk and Schmidt, (1) except that for the purpose of carrying out hydrogen ion activity measurements at 0 the calomel electrode, potassium chloride solution, and the hydrogen electrode vessel were immersed in an ice-water bath. A modified electrode vessel suitable for immersion in water was used. Uniform temperature was obtained by rapid stirring. For the purpose of determining the value of the calomel electrode at 0 the electromotive force of a hydrogen electrode immersed in a 0.01 N hydrochloric acid plus 0.09 N potassium chloride solution was determined. The relation between the measured electromotive force, the electromotive force of the calomel cell, and the hydrogen ion activity at 0 is given by the equation 1 Eo = Eo - log (H+). * Aided by a grant from the Chemical Foundation, Incorporated, and the Research Board of the University of California. We are indebted to the Cyrus M. Warren Fund of the American Academy of Arts and Sciences for the loan of the type K potentiometer. 285

2 286 Amino Acid Dissociation Constants where E. = the measured E.M.F. at O, Eo = the E.M.F. assigned to the calomel cell at 0 when (H+)o = 1 and (H+)o = the hydrogen c 6o 0.- z -5 y, d I 0 g45 - t t \ TITRATION CURVES OF ARGININE o = Arginine Monochloride *I x = Arginine Monochloride 2 } A =Water Blank Curve 25 C -m-e= Corrected Titration Curve 25 C CI = Arginine Monochloride I \ o.04m 0 C M 2 5 C. I, 5 6 PaH FIG. 1. I a LPCalc. ion activity at 0. The value of (H+)o was computed from the equat,ion (H+)o = Cro

3 Schmidt, Kirk, and Appleman 287 where C = concentration and yo = the activity coefficient of the hydrogen ion at 0. The value of yo was taken as (2). The value of Eo was found to be volt. The dissociation constant for water at 0 was taken as X lo g 60- *2= Y) ci 50-0 E 45- k n_4o- -0 -g 35- u ; j 25- b zo- 5 = 15- Z 6 lo- + o 5- cj 0 TITRATION CURVES OF LYSINE o=lysine Dichloride o.04m 25 C. a=water Blank Curve 25 C. a = Lysine Die hloride P H FIQ. 2. Arginine monochloride and lysine dichloride were isolated by the method of electrical transport described by Foster and Schmidt (3) and more recently elaborated by Cox, King, and Berg (4). A second preparation was obtained from the Eastman Kodak

4 288 Amino Acid Dissociation Constants TABLE I. Titration Data for Aspartic Acid, Glutamic Acid, and Histidine Dichloride at 0. All amino acid solutions were 0.04 M i N NaOH HCl Amino acid., c, f E.M.F. PaH z A! Aspartic acid. Glutamic acid. Histidine dichloride. 3l&ion cc E solution cc aolt Apparent dissociation constants. K,, = 1.7 X 10-d K,, = 4.9 x lo- 1 K,,, = 1.17 X 10- s 2.89 K,, = 7.25 X 10-s K,, = 6.0 X lo- K F,, = 1.95 X lo-l3 K, = 1.78 x K b, = 3.63 X lo-$ K$ = 9.12 X lo-14 PI Company. All products were recrystallized two to three times. Amino nitrogen determinations gave within the limits of error theoretical values. The glutamic acid and the histidine dichloride

5 Schmidt, Kirk, and Appleman 289 were the identical products which have been previously described (1, 5). The aspartic acid was a Kahlbaum preparation. It was recrystallized three times. The titration curve of arginine at 25 is shown in Fig. 1 and that of lysine at the same temperature is represented in Fig. 2. For arginine the following values were obtained for the dissocia,- tion constants at 25 : K,, = 3.32 X 10-13, K b, = 1.10 X 10-5, K b, = 1.05 X lo-12, and p1 = The values for lysine at this temperature are: K,, = 2.95 X lo-11, K t,, = 0.89 X 10W5, K t,, = 1.52 X 10-12, and p1 = Although the complete titration curves of arginine and of lysine at 0 are not shown a sufficient number of experimental points are given in each figure so that the apparent dissociation constants of the respective amino acid at this temperature may be calculated. On the basis of the plotted points shown in Fig. 1 the apparent dissociation constants of arginine at 0 are: K,, = 4.9 X 10-14, K 4 = 7.09 X lo- j, K b, = 2 X 10-13, and p1 = The apparent dissociation constants of lysine at 0 calculated from Fig. 2 are: K,, = 4.9 X lo- * K = 7.4 x 10-6, K a, = 1.82 X 10-13, and p1 = Tabl; I iives the experimental data from which the apparent dissociation constants of aspartic acid, glutamic acid, and histidine at 0 (also given in the table) have been calculated. Our values for the dissociation constants of lysine at 25 agree quite well with those which have been reported by Simms (6). He found K, = 3.4 X 10-11, I< b, = 1.11 X 10-5, and K b, = 1.11 X lo- 2. In the case of arginine the agreement with the values for the constants at 25 found by Simms (6) is not very good.1 The following are his recalculated values: K, = 2.29 X lo+, K b, = 1.43 X lo+, and K b, = 1.97 X The discrepancy lies in the values for K, and K/t,,. Our values for arginine are in better agreement with the data reported by Hunter and Borsook (7). Their recalculated data at 20 (8) yield the following values: K, = 1.4 x lo-13 K = 8.8 X 10-6, and K t,, = 1.07 X lo+. A comparison of (he Alues for the dissociation constants at 0 and 25 shows that the temperature influence is greatest on t,he smaller apparent dissociation constant values and least on the 1 In a private communication dated March 20, 1930, Dr. Simms states that his published values for the apparent dissociation constants of arginine at 25 are in error and accepts the values reported by us in this paper.

6 290 Amino Acid Dissociation Constants larger values. This is in agreement with the well known fact that the relative effect of temperature on the dissociation constants of highly ionized compounds is less than on compounds which are dissociated to a slight extent. Using the data for the dissociation constants at 0 and 25 of the amino acids given in Table II it is possible to calculate their apparent heats of ionization. Data for glycine, alanine, glycylglycine and glycylalanine obtained by this method of calculation have recently been published by Branch and Miyamoto (9). Meyerhof (10) had previously determined the heat of ionization of glycine and Adair, Cordero; and Shen (11) have reported the heat of ionization of hemoglobin. For the purpose of calculating the apparent heat of ionization of the amino acids reported in this paper we have made use of the equation -AH = d(rlnk) d log K d(i) = d(k) where T = the absolute temperature, R = the gas constant in calories, K = the true dissociation constant, and AH = the heat of ionization or the change in heat content of the solution as the result of ionization. In using this equation the following assumptions are made: (a) that the equation holds when the values for the apparent dissociation constants of the amino acids are used instead of the true dissociation constants; (6) that the increment AH is constant over the temperature range of O-25. This is equivalent to assuming that A log K d log K -=- between 0 and 25. These assumptions are the same as those which were made by Branch and Miyamoto (9). Although the validity of these assumptions is open to question the errors which are thus introduced are probably not much greater than certain other errors inherent in obtaining the values for the apparent dissociation constants.

7 Schmidt, Kirk, and Appleman 291 TABLE Apparent Heats of Zonization. II. Amino acid. remperature PK PK (o*) - pk (25 ) AH C. calories Arginine. 0 pk, = pk, = pk a, = pk b, = pk b2 = pk b9 = Histidine. Lysine. Aspartic acid. 0 pk, = pk, = PK a, = pk bl = PK b, = pk b2 = pk, = pk, = PK a, = pk b, = PK b, = PK b, = pk,, = PK., = ~K az = pk,* = PK b = pk b = Glutamic acid. 0 pk + = pkui = pk ap = pk,* = PKb = PKb = The values for the apparent heats of ionization AH of the amino acids are given in Table II. In the calculations we have used the values for the constants of arginine, lysine, histidine, aspartic acid, and glutamic acid experimentally determined by us and reported

8 292 Amino Acid Dissociation Constants in this paper. The constants for histidine at 25 were those previously reported by ourselves (5). The glutamic acid constants used were those which were determined by Simms (6, 8). If the constants for glutamic acid at 25 reported by Kirk and Schmidt (1) are used instead of Simms values an irrational value for AH' corresponding to pk,, is obtained. This is due to the fact that the value for AH corresponding to K, is small and a small error in the value for K,, is magnified in t,he value for AH'. On the ot.her hand, the value for AH' of glutamic acid corresponding to pk b, when similarly calculated are approximately 10 per cent greater than the value given in Table II This probably represents the error inherent in determining the values for AH'. The dissociation constants for aspartic acid at 3O were those which have been reported by Levene and Simms (12). An inspection of Table II shows that the order of magnitude of the values for AH' corresponding to K',, and K'q of arginine and of lysine and K',, and K'b, of aspartic acid and of glutamic acid are approximately the same and are not very different from the values for AH' reported for glycine and for alanine by Branch and Miyamoto (9). The values for AH' corresponding to K',, of aspartic acid and of glutamic acid and to K'b, of arginine and of lysine are small and roughly of the same order of magnitude. The value for K/t,, of histidine occupies a position intermediate between the values for K'4 of arginine and its own value for K'q and this is reflected in the value for AH' corresponding to K b, of histidine which is intermediate between the values for AH corresponding to K'b, of arginine and to K'a, of histidine. SUMMARY. 1. The apparent dissociation constants at 25 have been determined for arginine and for lysine and at 0 for arginine, histidine, lysine, aspartic acid, and glutamic acid. 2. On the basis of these data and certain others cited in the text the apparent heats of ionization of arginine, histidine, lysine, aspartic acid, and glutamic acid have been calculated. BIBLIOGRAPHY. 1. Kirk, P. L., and Schmidt, C. L. A., J. Biol. Chem., 81,237 (1929). 2. Scatchard, G., J. Am. Chem. Sot., 47,641 (1925).

9 Schmidt, Kirk, and Appleman 3. Foster, G. L., and Schmidt, C. L. A., J. Biol. Chem., 56,545 (1923). 4. Cox, G. J., King, H., and Berg, C. P., J. Biol. Chem., 81,755 (1929). 5. Schmidt, C. L. A., Appleman, W. K., and Kirk, P. L. J. Biol. Chem., 86,137 (192930). 6. Simms, H. S., J. Gen. Physiol., 11,629 (1928); 12,231 (1928). 7. Hunter, A., and Borsook, H., Biochem. J., 18,883 (1924). 8. Kirk, P. L., and Schmidt, C. L. A., Univ. Calij. Pub. Physiol., 7, 57 (1929). 9. Branch, G. E. K., and Miyamoto, S., J. Am. Chem. Sot., 52,863 (1930). 10. Meyerhof, O., Arch. ges. Physiol., 196, 22 (1922). 11. Adair, G. S., Cordero, N., and Shen, T. C., J. Physiol., 67,288 (1929). 12. Levene, P. A., and Simms, H. S., J. Biol. Chem., 56,801 (1923).

10 THE APPARENT DISSOCIATION CONSTANTS OF ARGININE AND OF LYSINE AND THE APPARENT HEATS OF IONIZATION OF CERTAIN AMINO ACIDS Carl L. A. Schmidt, Paul L. Kirk and W. K. Appleman J. Biol. Chem. 1930, 88: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1

THE EFFECT OF TEMPERATURE ON THE TITRATION CURVE OF CASEIN

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