STUDIES OF THE PEPTIDES OF TRIVALENT AMINO ACIDS

III. THE APPARENT DISSOCIATION CONSTANTS, FREE ENERGY CHANGES, AND HEATS OF IONIZATION OF PEPTIDES IN- VOLVING ARGININE, HISTIDINE, LYSINE, TYROSINE, AND ASPARTIC AND GLUTAMIC ACIDS, AND THE BEHAVIOR OF LYSINE PEPTIDES TOWARD NITROUS ACID*

BY JESSE P. GREENSTEIN

(From the Division of Biochemistry, University of California Medical School, Berkeley)

(Received for publication, March 22, 1933)

INTRODUCTION

In the study of complex biological systems it is oft.en desirable that their behavior be studied by comparison with the analogous behavior of simpler models of known structure and composition. In the case of the proteins, evidence collected on their simplest units, the amino acids and their peptide combinations, helps to elucidate certain characteristics of the larger molecules.1 System- atic data are being collected in t,his laboratory (3-6) on the electrochemical behavior of the amino acids, which in many re- spects parallels the amphoteric behavior of the natural protein. In the present series of st.udies (7, S), the attempt is made to compare the dissociation values found in the prot,eins with the values deter-

* Aided by a grant from The Chemical Foundation, Inc., and the Re- search Board of the University of California to Professor Carl L. A. Sch- midt. 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.

i The compounds described in this paper, with the exception of glycyl- proline, were prepared by the author while National Research Council Fellow in Biochemistry at the Kaiser Wilhelm Institute, Dresden, 1931- 32. The peptide, glycylproline, was kindly furnished by Professor M. Bergmann of the above Institute.

1 For an extensive discussion and summary of this problem, see Cohn

(1, a. 603

604 Peptides of Trivalent Amino Acids. III

mined from synthetic peptides containing those free groups not involved in peptide linkage, and responsible for the protein-ionizing capacity. Such groups are the e-amino group of lysine, the imida- zole ring in histidine, the free carboxyl group in the dibasic aspartic and glutamic acids, the guanidine nucleus of arginine, and the hy- droxyphenyl ring of tyrosine. Alterations in the values of the dis- sociation constants of these groups when found in the amino acids are decidedly marked when these acids are combined in peptide link- age. Further lengthening of the chain beyond the dipeptide stage, as Stiasny and Scotti (9) indicated, produces only very much smaller changes in the values of the amino and the carboxyl groups. If the protein therefore is to be considered as composed largely of long peptide chains, and if any forces other than those due to primary valency are neglected, the protein dissociation values may best be compared with those found in the dipeptides. The mutual effect of the free ionizing groups crowded together in the smaller synthetic molecules will reflect the analogous behavior of such groups in the protein.

In previous papers (7,8) this effect was studied in such peptides as histidylhistidine, aspartylaspartic acid, and tyrosyltryosine. It was found that, while the interposition of the peptide linkage has always the effect of considerably weakening the amino and the carboxyl groups, this weakening may be somewhat modified by the presence of other free ionizing groups in the molecule. The shifts in the values of these groups are less than those of the amino or the carboxyl group, and their direction toward more acid or alkaline range will depend upon the nature of the groups in proxim- it,y. Thus the basic imidazole group will be slightly more acid than in histidine when found next to the basic amino group in the peptide (7); on the other hand, the acid hydroxyphenyl ring in the peptide, when the carboxyl group is in proximity, will be slightly more alkaline than in tyrosine (8). These general phenomena are already familiar from the work of Ostwald (10) and Bredig (11) and are further exemplified in the results on the diverse substances described below. In general, it may be stated that the effect of these extra ionizing groups is to increase the dissociation of the carboxyl group whether in the amino acid or in the peptide. The acidic property of the carboxyl group in tyrosine, histidine, and arginine is uniformly higher than in glycine (1) and this strengthen-

J. P. Greenstein 605

ing effect, combatted by the weakening influence of the peptide linkage, is carried over into the peptide molecule. Not only the groups peculiar to these acids, but the presence of additional amino groups will likewise cause an increased acid dissociation, and, as shown by the author (12) and by Schmidt, Appleman, and Kirk (5), this increase will be inversely proportional to the distance between the dissociating groups.

In the present communication, the dissociation values of pep- tides containing several free groups will be reported, as well as their free energy changes and heats of ionization. Data on the heats of ionization of several complex amino acids have been obtained by Schmidt, Kirk, and Appleman (4) and by Branch and Miyamoto (13). Miyamoto and Schmidt (6) have reported the apparent free energy and entropy changes due to ionization of the amino acids. The present study is concerned partly with the dissocia- tion of peptides containing the guanidine nucleus, the oxyphenyl and imidazole rings, and the free amino and carboxyl groups, as reflected in the titrations of phenylalanylarginine, tyrosylarginine, aspartyltyrosine, histidylglycine, lysylglutamic acid, and lysyl- lysine. For purposes of comparison the titrations of glycylglycine and of phenylalanylglycine have been redetermined and included. Potentiometric measurements were made at 25” and at 0’ and the heats of ionization were calculated from the results obtained at these temperatures. While all the peptides hitherto studied con- tain the usual substituted acid amide binding between amino acids, the peptide, glycylproline, is unique in that its peptide linkage con- tains no hydrogen. It was thought to be of interest to include the titration of the pept.ide of this interesting and widely occurring imino acid.

It has been shown by Dunn and Schmidt (14) that the rate of deamination of variously substituted amino acids with nitrous acid decreases with increasing distance between the amino and the carboxyl groups, and the same authors and Schmidt (15) have demonstrated that this rate is likewise a function of the tempera- ture. In the present paper will be reported the rate of deamination of peptides of lysine as reflected in the behavior at constant tem- perature of lysyllysine, lysylglutamic acid, and lysylhistidine to- ward nitrous acid. It will be shown that this rate is about double that characteristic of lysine.

606 Peptides of Trivalent Amino Acids. III

EXPERIMENTAL

Preparation of Materials--The peptides, phenylalanylarginine, t’yrosylarginine, and histidylglycine, were prepared respectively according to the general azlactone methods of Bergmann and Kijst,er (16), Bergmann, Zervas, and du Vigneaud (17), and Berg- mann and Zervas (18). The arginine for the synthesis of the arginine-cont.aining peptides had been isolated from gelatin by the benzaldehyde procedure of Bergmann and Zervas (19). Aspartyl- tyrosine was prepared according to the new carbobenzoxy method of Bergmann and Zervas (20). Lysylglutamic acid and lysylhis- t,idine were identical samples reported by Bergmann, Zervas, and Greenstein (21). Lysine anhydride, phenylalanylglycine, and glycylglycine were prepared respectively according to the methods of Fischer and Suzuki (22), Fischer and Blank (23), and Fischer and Forneau (24). The lysine dihydrochloride employed for the deter- mination of the rate of deamination by nitrous acid was isolated from hemoglobin after the procedure of Vickery and Leavenworth (25). All of these substances had been recrystallized and their melting points and elementary analyses checked. Glycylproline, supplied through the kindness of Professor Max Bergmann, was part of the identical sample reported by Bergmann, Zervas, Leinert, and Schleich (26).

Procedure

In nearly all cases a 0.02 M solution of the peptide in water was prepared, appropriate aliquots were removed and treated with either 0.040 M or 0.080 M HCl or NaOH, and the resulting solution was made up to such volume that the end-concentration of ampho- lyte was 0.01 M. For the preparation of a 0.02 M solution of lysyl- lysine from lysine anhydride dihydrochloride, a sample of 0.1645 gm. of the latter substance was dissolved in 7.5 cc. of N NaOH and allowed to stand for 24 hours at room temperature. At the expira- tion of this time, 7.5 cc. of N HCl were added, and the mixture was made up to 25 cc. with water. As shown previously (7, S), the diketopiperazine is quantitatively split under these conditions and this is further confirmed in the case of this substance by the amino nitrogen and the t,itration data.

Potentiometric readings were made at 25” and at O”, the hy- drogen and calomel electrodes being kept at the former tempera-

J. P. Greenstein 607

ture by a controlled air chamber, and at the latter temperature by an ice water bath. The 0.1 N KC1 calomel electrode was standard- ized at 25” with 0.1 N HCl, Scatchard’s value for -&+ of 0.841 being used (27). For the determination of the value of the calomel electrode at 0”, a solution of 0.01 N HCI plus 0.09 N KC1 was em- ployed. The value of y n+ of this solution was taken as 0.907 (27). The negative logarithm of the dissociation constant of water was accepted at 25” as 13.998 and at 0” as 14.943. The activity

aj 1.4

g m 1.2

& 1.0

z 0.8

a 0.6 + O 0.4 Y,

2 0.2

0 2 4 6 8 1012

PaH FIQ. 1. Titration curve of phenylalanylarginine based on the constants:

pK,’ = 2.66, pK2’ = 7.57, pK;’ = 12.40. o indicates experimental points at 25” and 0 at 0’.

coefficients of H+ and OH- employed for the calculation of the acid and the base bound by the ampholyte were assumed identical with those with respect to pure HCl and NaOH solutions re- spectively, and were taken in part from the data of Lewis and Ran- dall (28) and in part from the results of blanks carried out with pure water.

For the purpose of illustration, the titration curve of phenyl- alanylarginine is given in Fig. 1. The ordinate represents the acid or base bound per mol of peptide, the abscissa the puH. The

608 Peptides of Trivalent Amino Acids. III

experimental points are indicated at 25” by circles and at 0” by squares. The curve is theoretical and is calculated from the titration constants chosen.

The dissociation of each free group is represented by CY in the mass action expression for the behavior of a monovalent acid or base :

G’ = H @/(l-a)) or pG’ = paH + log ((l-a)/~()

where G’ is the apparent titration constant2 related to the classical apparent dissociation constant K’ by expressions first developed by Simms (29). Inasmuch as the corresponding values of G’ and K’ for each group differ from each other by an amount less than the experimental error in determining such constants, it seemed preferable to retain only the value for K’ throughout this communi- cation

The apparent dissociation constant pIC’ of each isolated group ma,y then be calculated from the pnE-I at which that group is half neutralized. When, however, two or more groups dissociate simul- taneously within the same paH range, then the pK’ values of these groups must be so chosen that at each paH value that the experi- ments reveal, the sum of the dissociation stages, zcu of all such groups, will yield the mols of acid or base combined; Zar = X.~

For the calculation of the free energy changes due to ionization, the following relation, similar to that employed by Miyamoto and Schmidt (6), was used (AW)’ = -IiT In K’ where (AP”)’ is a con- stant at any one temperature and R’ is the apparent dissociation constant. At constant temperature, the change in free energy is a measure of the maximum work performed by the system between the standard and the actual states. In the above expression, the change in free energy represents the work involved in each step of ionization (represented by K’) and will be proportional to the algebraic sum of the various effects due to the nature of the dis- sociating group, the influence of other substituents upon this group, and t,he electrostatic forces existing among all other groups in the

2 Inasmuch as in the above equation the first member on the right-hand side is expressed as activity and the second member as concentration, the constant pG’ is of a hybrid or apparent character.

3 For definition of LY, the acid or base combined per mol of ampholyte, refer to Paper I of this series (7).

J. P. Greenstein 609

molecule. These effects have been estimated by Bjerrum (30) and by Simms (31) in the case of the isomeric divalent acids. It is doubtful whether similar considerations could be applied to the complex ampholytes, and the values of (APO) listed in Table I are limited to the actual numerical values found for K’. Miya- moto and Schmidt (6) have shown that no appreciable error was introduced into the calculations of the free energy when the values for the apparent dissociation constants were used in place of the true dissociation constants.4

For the purpose of calculating t.he apparent heats of ionization, the equation

d(R In K’) -(AH) = d (1,T) = 14,940 APR’

was employed, according to the assumptions previously made by Schmidt, Kirk, and Appleman (4) and by Branch and Miyamoto (13).

Amino Nitrogen Determinations-The usual Van Slyke apparatus was employed in a room whose temperature varied from 22” by not more than fl”. The solutions of lysine and of the peptides were so prepared that a given aliquot of 2 cc. would yield about 2 cc. of nitrogen gas. Time intervals of shaking from 1 minute in duration upwards were employed. Customary blank deter- minations were made.

DISCUSSION

In Table I are listed the dissociation values and heats of ioniza- tion of the peptides studied as well as some data on amino acids reported by Schmidt, Kirk, and Appleman (4).

It is apparent that the free groups of the peptides, while usually dissociating at ranges far removed from those of the same groups in amino acids, nevertheless yield similar values for the heat of

4 The transformation of K’, the so called apparent constant, to K, the thermodynamically defined constant when all members in the reaction at equilibrium are expressed as activities, consists essentially in multiplying K' by the ratio of the activity coefficients of the charged and uncharged forms of the ampholyte. For most practical purposes, the activity coeffi- cient of the non-ionized molecule may be taken as unity. The activity co- efficients of certain amino acid ions derived from freezing point data have been reported by Hoskins, Randall, and Schmidt (32).

610 Peptides of Trivalent Amino Acids. III

ionization. That strongly acid groups have very small heats of ionization, whereas strongly basic groups possess large heat values in the neighborhood of 10,000 to 12,000 calories, has already been pointed out among others by Kolthoff (33), Ebert (34), and Meyer- hof (35).6 The question of the influence of temperature on dis- sociation and the zwitter ion constitution of amphoteric electro- lytes has been summarized by Cohn (1). It has been shown by Schmidt, Kirk, and Appleman (4) that the strongly basic groups such as the amino and guanidine groups possess values of heat of ionization in the neighborhood of 10,000 to 12,000 calories, whereas the weakly basic imidazole ring in histidine yields a value con- siderably lower than this. Similar values are obtained with peptides containing these groups (Table I). Again, the strongly acid carboxyl group has a negligible heat of ionization both in the amino acids and in the peptides, whereas the very weakly acid hydroxyphenyl ring yields a value intermediate between those of full basic and full acid heat values, namely in the neigborhood of 6000 calories (Table I). It is probable, therefore, that the dissoci- ation of the same groups in the larger protein molecule will behave similarly with temperature and some evidence in this direction has been presented by Pertzoff and Carpenter (36).

The values of the free energy change (APO)‘, corresponding to each step in ionization described by K’, will be proportional to the actual numerical value of K’. Consequently, these values will be somewhat different, in the peptides and in the amino acids. The work involved in the ionization of the carboxyl groups of the monoaminomonocarboxylic acids, as shown by Miyamoto and Schmidt (6), is of the order of 3000 calories, while that of the amino groups of the same acids is about 13,000 calories. An intermediate value is obtained for the imidazole ring of histidine, and the highest value of 17,000 calories is given by the guanidine

5 It is therefore, possible, by estimating the magnitude of the apparent heat of ionization, to predict the nature of the group which is dissociating. This is clearly of advantage in dealing with the complex peptides contain- ing crowded free groups which may have, as in the case of tyrosylarginine, several groups simultaneously dissociating within a rather narrow pH range. Those organic radicals which are either weakly acid or weakly basic, such as the hydroxyphenyl and the imidaaole rings respectively, will yield intermediate heat values and by this criterion may be readily identified.

J. P. Greenstein 611

nucleus of arginine. In the case of the peptides (Table I) the work involved in the ionization of a carboxyl group will be slightly greater, that of an amino group will be considerably less, than the work involved in the ionization of the same groups in the amino acids. The extent of these differences will vary with the nature of other substituents in the molecule. When extra basic substituents are present, the work involved in the carboxyl dissociation will be still less than when no such groups are present. In the case of lysyllysine, this energy will be even less than that involved in the ionization of the carboxyl group in lysine (Table I).

In a consideration of the free energy change as a function of the temperature, and if the Gibbs-Helmholtz equation is employed

(AP)’ = (AH)’ + T (d (AP)‘/dT)

it is evident that in the case of the carboxyl group dissociation where (AH)’ is negligible or zero, the free energy change will be proportional to the absolute temperature. On inspection of the values for (APO) corresponding to the carboxyl groups and listed in Table I, it is observed that these values increase by 300 to 400 calories between O-25’. Furthermore, if the assumption be made that the change in free energy varies linearly with the temperature, values of (APO) of the order of magnitude of 3000 to 4000 calories are obtained, which are characteristic of the free energy change involved in the ionization of the carboxyl group. Conversely, values for (AH)’ of 500 to 1000 calories may be calculated from the known values of (AFO)’ and its temperature coefficient for the carboxyl group.6 On the other hand, when the change in free energy is independent of the temperature, that is when d (AFO)‘/dT = 0, then the change in free energy due to ionization should equal the heat of ionization. This relation is closely followed in the case of the amino and imidazole groups of all the compounds listed in Table I. The values of (AFO)’ corresponding to the imidazole and amino groups are practically constant between O-25” and according to the above equation the values of (AFO)’ and (AH)’

6 Since (AH)’ values are proportional to the difference in the values of pK’, any errors in the determination of the latter may be magnified in the values of (AH)‘. An error in ApK’ of 0.05 unit will cause a deviation in (AH)’ of 750 calories. In contrast, an error in pK’ of 0.05 unit will cause an error in (AP) of about 65 calories.

612 Peptides of Trivalent Amino Acids. III

TABLE I

Ionization and Energy Values of Peptides

Phenylalanylargi- nine

Arginine (Schmidt et al.1

Phenylalanylgly- cinet

Tyrosylarginine

Aspartyltyrosine

Histidylglycine

Histidine (Schmidt et al.1

Glycylglycinej

--

-

“C.

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

0 25

Group*

Carboxyl

Amino

Guanidine

“

Carboxyl

Amino

Carboxyl

Amino

OxyphenyI

Guanidine

Carboxyl

“

Amino

Oxyphenyl

Carboxyl

Imidazole

Amino

Imidazole

Carboxyl

Amino

PK’ ApK’ (AH)’ (AFO)’ p1

-~ __~- calories calories

2.69 3,300 10.72 2.66 0.03 450 3,700 9.99 8.25 10,300 7.57 0.68 10,150 10,300

13.20 16,600 12.40 0.80 11,95017,000 13.31 16,700 11.55 12.48 0.83 12,40017,100 10.76

3.14 3,900 5.76 3.10 0.04 680 4,300 5.40 8.38 10,500 7.71 0.67 10,000 10,500 2.65 3,300 8.93 2.65 0 0 3,600 8.38 8.09 10 ) 100 7.39 0.70 10,500 10,100 9.76 12,200 9.36 0.40 6,000 12,800

12.50 15,700 11.62 0.88 13,00015,900

2.18 2,700 2.88 2.13 0.05 750 3,000 2.85 3.57 4,500 3.57 0 0 4,900 9.60 12,100 8.92 0.68 10,160 12,200

10.65 13,400 10.23 0.42 6,200 14,000

2.42 3,000 7.42 2.40 0.02 300 3,300 6.81 6.30 7,900 5.80 0.50 7,500 7,900 8.54 10,700 7.82 0.72 10,800 10,700 6.50 8,100 8.13 6.04 0.46 6,900 8,300 7.58 3.16 3,900 6.00 3.12 0.04 680 4,300 5.65 8.86 11,100 8.17 0.69 10,300 11,200

Lysylglutamic acid

Lysyllysine

Lysine (Schmidt et al.)

Glycylproline

J. P. Greenstein

TABLE I-Concluded

613

Tern- pera- ture

"C.

0 25 0

25 0

25 0

25 0

25 0

25 0

25 0

25 0

25 0

25 0

25 25

Group’ PK' ApK' (AH)' (AFo)' PI

Carboxyl

“

Amino

“

Carboxyl

Amino

‘I

‘I

Carboxyl

Amino

I‘

Carboxyl Amino

----- cakwies caltie.

2.98 3,700 6.45 2.93 0.05 750 4,000 6.10 4.47 5,600 4.47 0 0 6,100 8.45 10,600 7.75 0.70 10,50010,600

11.30 14,200 10.50 0.80 11,950 14,400 2.10 2,600 10.87 1.95 0.15 2,000 2,700 10.04 9.02 11,300 8.17 0.85 12,700 11,200

10.21 12,800 9.45 0.76 11,350 12,900

11.52 14,500 10.63 0.89 13,30014,600 2.20 2,700 10.56 2.18 0.02 300 3,000 9.74 9.81 12,300 8.95 0.86 12,800 12,300

11.31 14,200 10.53 0.78 11,60014,400 2.84 3,900 5.73 8.62 11,800

* The constants calculated are assigned to specific groups on the basis of the zwitter ion constitution of amphoteric electrolytes.

t Values at 25” for phenylalanylglycine were reported by Mitchell and Greenstein (37) as pK,’ = 2.05, pK2’ = 8.02. It is believed that these re- sults are in error owing to the use of impure material. These investigators reported an erratic potential (p. 259) which was undoubtedly due to the presence of cinnamoyl derivatives and which would account for the elec- trode poisoning described. After several recrystallizations of the peptide from hot water, no difficulties were encountered.

$ Dissociation values for glycylglycine at these two temperatures are comparable with those determined by Branch and Miyamoto for this com- pound (13). They found pK,’ = 3.02 at 0” and pK,’ = 3.06 at 25”, with (AH)’ = -600 calories. The basic constant was reported as pK2’ = 8.80 at 0” and 8.13 at 25” with (AH)’ = 10,000 calories.

614 Peptides of Trivalent Amino Acids. III

should be about equal. Within the experimental error, these values may be considered equal (Table I).’

The temperature coefficient of (APO) in the case of the carboxyl, hydroxyphenyl, and guanidine groups, is always positive and of the same magnitude. The behavior of the latter two groups can- not be described in terms of the Gibbs-Helmholtz equation. It is possible that in the instance of the guanidine group the very con- siderable errors resulting in the determination of its dissociation constant in regions of high alkalinity are a contributing factor.

While the heat of ionization appears to be cha,racteristic of the nature of the dissociating group and independent of the constitu- tion of the ampholyte molecule, the value of the titration constant relating to any particular group is a function both of that group’s position in the molecule and the presence of adjoining groups. In all cases, the effect of the introduction of the peptide linkage be- tween amino acids is simultaneously to weaken and narrow the range of dissociation of the free groups over that of the same groups when found in the amino acids. Modifying this weakening effect of the acid amide linkage on the carboxyl and amino groups is the influence of neighboring substituted radicals such as the imidazole, guanidine, and oxyphenyl groups. The change in the values of these latter groups in the peptide molecule over that in the amino acids is usually quite small. On the other hand, the alteration in the dissociation values of amino and carboxyl groups is quite large and is the resultant both of the influence of the peptide linkage and the nature of the substituted group or groups in proximity.

The presence of basic radicals in the molecule often results in a shift of the isoelectric point toward a lower acidity. For example, peptides like phenylalanylarginine, histidylglycine, and histidyl- histidine, containing the guanidine or imidazole groups, possess isoelectric points more acid than those of the free arginine or his- tidine molecules (7). This shift is reflected in the simultaneous increased acid strength of the carboxyl group and decrease in strength of the amino group which become clear on comparing

7 While the validity of using the modified Gibbs-Helmholtz equation applied to the data presented may be questionable, owing to the numerous simplifying assumptions made in the calculations of (AFO)’ and (AH)‘, it is believed that it is capable of an approximate description of the phenomena presented.

J. P. Greenstein 615

these values in this type of complex peptide with those of glycyl- glycine (Table I). The presence of the guanidine nucleus in argi- nine and that of the imidazole ring in histidine result in this same type of acid shift over the values characteristic for the monoamino- monocarboxylic acids. Thus glycine possesses a pK’ value for the carboxyl group of 2.33 (l), arginine, however, one of 2.01 (4), and histidine 1.82 (4). The carboxyl dissociation value in glycyl- glycine is 3.12, that of phenylalanylarginine is 2.66, and of histidyl- glycine 2.40.

The intensified acidity of the carboxyl group due to basic sub- stitutents is, however, best illustrated by lysyllysine. This sub- stance, containing three amino groups and one carboxyl group, may be considered as differing from lysylglutamic acid in that the former possesses an amino in place of a carboxyl group. The effect on the dissociation of the carboxyl group in lysyllysine due to the presence of this third amino group is very marked. The pK’ value for the carboxyl group in lysylglutamic acid is 2.95; in lysyllysine it is 1.95. The corresponding dissociation value for the same group in lysine is 2.18. The customary weakening effect of the peptide linkage on the carboxyl group is more than coun- teracted by the presence of several amino groups and this phen- omenal shift may be compared with the dissociation value found by the author (12) of 1.33 for the carboxyl group in 1y, p-diamino- propionic acid. In the latter substance there is likewise achieved a great concentration of amino groups within a rather small molecu- lar area. The peptide, histidylhistidine, which like lysyllysine also contains three basic groups and one carboxyl group, yields a low pK’ value for the latter group of 2.25 (7).

Another type of substituent whose introduction into the peptide molecule results in a shift toward more acid values is the weakly acid hydroxyphenyl group. In tyrosine it leads to an increase in acid dissociation and a decrease in basic dissociation as compared with glycine (1). In tyrosyltyrosine and glycyltyrosine (8) it causes a slight shift toward a more acid isoelectric point than that of tyrosine. In tyrosylarginine it shifts the isoelectric point from 9.99 in phenylalanylarginine to a value of 8.38. The value of p1 in aspartyltyrosine is slightly more acid than that of aspartyl- aspartic acid (7).

Lysylglutamic acid is the first peptide prepared of a diamino-

616 Peptides of Trivalent Amino Acids. III

monocarboxylic acid and a monoaminodicarboxylic acid. Its properties should not be very different from those of glycylglycine and in point of fact its isoelectric point of 6.10 at 25’ is only slightly higher than that of glycylglycine, namely 5.65, at the same tem- perature. Furthermore, the temperature coefficient of the iso- electric point of bothcompounds betweeno-25’is 0.35 unit (TableI).

While amino and carboxyl groups are usually considerably weakened by the interposition of the peptide linkage, the shifts in the values of the substituted groups described above are small in comparison, and their direction is determined by the nature of the groups in proximity. It had been previously suggested that in histidylhistidine (7) the imidazole ring next to the amino group would be shifted to a more acid value than in histidine. Whereas in this peptide containing two imidazole rings it was difficult to assign a value to either ring, the results obtained now with histidyl- glycine confirm this assignment. In t,he latter peptide, the lone imidazole radical is located on the acyl amino acid and in p position to the amino group. Its pK’ value at 25” is 5.80 as compared with 6.04 in histidine (4) and 5.60 in histidylhistidine (7). Similarly it had been suggested by the author (8) that the shift in the dis- sociation value of the hydroxyphenyl group would be determined by the nearness of a carboxyl or an amino group. In glycyltyro- sine, where the oxyphenyl ring is next to the carboxyl group, its dissociation is weakened and is shifted to’s slightly more alkaline range. In tyrosyltyrosine, the value of the more acid pK’ for the radical was therefore assigned to that nearer the amino group. That this assignment was correct is shown by the dissociation values found in tyrosylarginine and in aspartyltyrosine. In the former, where the oxyphenyl group is on the acyl amino acid, its value is 9.35; in the latter compound it is found on the distal amino acid and its value is 10.20. These values are respectively more acid and more alkaline than that pK’ value of tyrosine, namely 10.07 (38). The dissociation values of the two rings in tyrosyltyrosine were 9.80 and 10.26, and the assignments previously made of these values, based on the structure of this compound, are confirmed by the results obtained in the present study.

In the case of the guanidine group, its shift toward a weaker value is slight; i.e., from 12.48 in arginine to 12.40 in phenylalanyl- arginine and to 11.62 in tyrosylarginine (Table I). The strength

J. P. Greenstein 617

of this group will probably be less affected by neighboring substitu- ents and will depend more on the number of peptide linkages in the chain. It is probable that arginine when in the acyl peptide position will yield a somewhat more alkaline value for the guanidine group than when found in the amino acid alone.

Glycylproline with its -CON= peptide linkage affords an in- teresting contrast with glycylglycine which possesses the -CONH- type of peptide binding. While the effect of both types of linkages is to weaken both amino and carboxyl groups as compared with the amino acid, the former linkage has a markedly less weakening influence than the latter. The dissociation values of the carboxyl groups are 2.84 and 3.12 in glycylproline and in glycylglycine re- spectively, with values of 8.62 and 8.17 for the respective amino groups. The isoelectric points of both compounds are practically identical, with 5.73 for glycylproline and 5.65 for glycylglycine. These results with glycylproline arc comparable with those ob- tained by Levene, Simms, and Pfaltz (39) in the case of glycyl- sarcosine. This compound, with a type of peptide linkage similar to that of glycylproline, yielded pK’ values for the carboxyl group of 2.83 and for the amino group of 8.54, with the isoelectric point at 5.68.

In Table II are gathered the data relating to the rates of deamina- tion of lysine, lysyllysine, lysylglutamic acid, and lysylhistidine. Whereas lysine requires about 15 minutes at ordinary temperature for the quantitative removal of the e-amino group, it is found here that peptides containing lysine are remarkable in that their total amino nitrogen is removed in about half this time at the same temperature. In a personal communication from Professor M. Bergmann, the author was informed that similar results had been found in the former’s laboratory. This rather rapid rate of de- amination of the lysine peptides stands in contrast with the much longer time required to deaminize a protein like gelatin (40) or casein (14), and points to a difference in the surface constitution of the larger and smaller molecules. These differences in consti- tution most probably do not affect the ionization of the various groups, since these depend more upon the nature and character of contiguous groups as well as upon the distance between them. The cu-aminomonocarboxylic acids possess different constitutions and the rate of introducing substituents, as shown by Abderhalden

618 Peptides of Trivalent Amino Acids. III

and collaborators (41-43) in the case of the conversion of the bromo derivatives of the isomeric valines and leucines into the amino acids and the betaines, will be clearly different in each case. How- ever, their dissociation values are all approximately equal. It is highly probable that the chemical reactivity of the free groups involved in the protein or the amino acids bears little if any re- lation to their ionizing capacity, and the differences in the rates of

TARLE II

Amino Nitrogen Liberated at 88’

Time

min. 1 2 3 4 5 6 6.5 7 8 9

10 11 12 14 15 16 18

Lysine

per cent

64

84 87

90 91

95 97 98 99

100 100 100

K’

0.17 0.15

0.14 0.13

0.13 0.14 0.14

-i- Lysyllysine

per cent 73 82 87 90 92 95 96

100 100 100

K’

0.29 0.25 0.22 0.21

-

-

- per cent

80 85 91 94 95

99 100 106 100 100

100

-

K’

0.27 0.26 0.25 0.22

Lysylhistidine

wt cent

63 84 90 92 96 98 99

100 100 100

K*

0.33 0.28 0.28 0.28

* K = constant for the monomolecular reaction. Its average value for lysine is 0.14, for lysyllysine 0.24, for lysylglutamic acid 0.25, and for lysyl- histidine 0.29. Dunn and Schmidt (14) report a value of K = 0.11 at 23” for the e-amino group of lysine.

deamination of the e-amino group in lysine, lysine peptides, and the protein, must be sought in causes arising from differences in chemical structure other than those concerned with alterations in ionization.

I wish to thank Professor Carl L. A. Schmidt for his advice and suggestions during the course of the titration experiments herein recorded.

J. P. Greenstein

SUMMARY

1. The apparent dissociation constants at 0” and at 25” have been determined and the free energy changes and heats of ioniza- tion have been calculated for phenylalanylarginine, tyrosylarginine, aspartyltyrosine, histidylglycine, phenylalanylglycine, glycyl- glycine, lysylglutamic acid, and lysyllysine.

2. It is shown that the heat of ionization of the carboxyl group is negligible, while that of the amino and guanidine groups is con- siderable, and of the order of 10,000 to 12,000 calories, whereas those heat values of the imidazole and oxyphenyl radicals possess intermediate magnitudes. Although the actual titration values of the free groups of the peptides are markedly different from those of the same groups when found in the amino acids, and may fur- thermore be influenced by the presence of other substituents, the values of the heats of ionization corresponding to these groups are apparently independent of the constitution of the molecule. In the case of peptides containing several free charges, the temperature coefficient thus permits some means of identification of the ionizing group.

3. The change in free energy due to ionization corresponding to the carboxyl group is slightly greater in the peptides than in the amino acids, and possesses an appreciable temperature coefficient. On the other hand, the free energy change of the amino groups in the peptides is considerably less than that of the same group in the amino acids and its value is practically independent of the tempera- ture. The thermodynamic behavior of both groups and of the imidazole radical may be described in terms of the modified Gibbs- Helmholtz equation. The temperature coefficient of the free energy change of the carboxyl, hydroxyphenyl, and guanidine groups is always positive and of the same order of magnitude.

4. While in the transition of amino acids to peptides the amino and carboxyl groups are considerably weakened, tfhis weakening may be modified by t,he presence of various groups such as the imidazole and oxyphenyl rings, and the guanidine nucleus. A concentration of basic groups as in lysyllysine may even more than overcome the weakening influence of the peptide linkage and cause the carboxyl group to dissociate at a reaction more acid than in lysine itself. The shifts in the values of these ionizing radicals

620 Peptides of Trivalent Amino Acids. III

are small in comparison to that undergone by amino and carboxyl groups, and their direction is determined by the position of these groups in the molecule.

5. The titration constants at 25” have been determined for gly- cylproline and compared with those values for glycylglycine. These compounds differ in the nature of the peptide bond. It was found that the weakening influence of the peptide linkage of the former compound on amino and carboxyl groups is considerably less than that of the latter. However, the isoelectric points of both sub- stances are practically identical.

6. The rates of deamination by means of nitrous acid of lysine, lysyllysine, lysylglutamic acid, and lysylhistidine have been de- termined. Whereas lysine requires approximately 15 minutes for complete deamination, t.he lysylpeptides all require about half this time.

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