ERICELLENBOGEN
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Vol. $3
[CONTRIBUTION FROM THE LABORATORIES OF THE DEPARTMENT OF BIOCHEMISTRY, COLUMBIA UNIVERSITYCOLLEGEOF PHYSICIANS AND SURGEONS, A S D THE DEPARTMENT OF BIOCHEMISTRY AND XUTRITIOY, GRADUATE SCHOOL O F P r R L I C HEALTH, UNIVERSITS O F PITTSBI’RGII]
Dissociation Constants of Peptides. I. A Survey of the Effect of Optical Configuration B Y ERICELLENBOGEN2f3 RECEIVED MARCH 27, 1952 The dissociation constants of a number of peptides containing glycine, alanine and lysine were determined in an attempt to study the effect of optical configuration of the amino acid residues on the dissociation of the ionizable groups. In general, replacement of an amino acid residue by its enantiomorph will result in small, but significant changes in the dissociation constants of such groups. As part of this study, revised values for the dissociation constants of lysine are proposed. An attempt was made to interpret the results obtained in terms of spatial configuration of the peptide molecules. It is also suggested that revised values be assigned to the dissociation constants in pi oteins of the a-carboxyl, or-amino and e-amino groups.
Recently, Brand and his co-workers4undertook a study of the optical rotatory power of amino acid residues in peptides. A large number of interesting peptides of known optical configuration and composition was synthesized, some of which became available to us for the determination of their dissociation constants. Experimental
manual. During titrations a stream of moist nitrogen was passed over the surface of the solutions to prevent absorption of atmospheric carbon dioxide. With the proper technique, no appreciable errors due to evaporation arc introduced. pK‘ values were calculated for each increment, using the well-known relation
pK‘ = PH - log (A)/(B) (1) where (A) and (B) are the concentrations of the acid and its conjugate base, pH is the measured quantity, and p l i ‘ = p K All peptides, with the exception of dilysine (LL), were part log ( Y A ) / ( ~ R ) . Since the initial concentration of the comof the same lots on which residue rotations were determined, pound to be titrated was known, it remained to determine and whose syntheses and analyses are described elsewhere.4,5 the concentrations of (A) and (B) after each addition of acid All reagents were of C.P. grade and were used without or base. This was accomplished by carrying out blank tifurther purification, with the exception of the sodium chlo- trations of the acid and base a t the same ionic strength, from ride which was fractionally crystallized. The first two which a set of “apparent” activity coefficients, 7 ’ ~ and batches were discarded, and the mother liquor of the third ?’OH- was obtained. These values were then employed in batch was ‘treated with concentrated hydrochloric acid. the evaluation of the quantities (A) and (B). The pH meter The precipitated sodium chloride was washed with absolute was frequently standardized against buffers of the following alcohol followed by ether, the crystals were dissolved in the known pH’s: 4.00, 7.00 and 10.00. For our calculations we minimum amount of boiling water, and the solution was assumed that UH+ = antilog (--pH). I t is assumed that allowed to cool. The resulting crop of crystals was dis- liquid junction and asymmetry potentials in this type of cell carded, and the mother liquor was treated with 6 N hydro; are accounted for in the set of “apparent” activity coeffichloiic acid. The material thus obtained was dried a t 140 cients, and that the relative magnitude of these potentials and was used as the source of neutral salt in all experiments. does not change upon addition of the peptides. Sodium hydroxide, approximately 0.100 N was prepared by Results diluting a carbonate-free solution of saturated sodium hydroxide with carbonate-free distilled water, and was standCalibration.-The technique was tested on a ardized frequently against Bureau of Standards potassium acid phthalate. Approximately 0.100 N hydrochloric acid few amino acids whose dissociation constants were was prepared by dilution of constant boiling acid, and served well known. The results obtained with glycine, as auxiliary standard. L-histidine, L-alanine and L-arginine are compared Titrations were carried out on the Beckman model G p H meter equipped with the external electrode assembly in Table I with those reported in the literature. (thirty-inch leads). The general purpose electrode was The table also includes the results obtained with employed for measurements in the pH region 2 to 8.5, and TABLE I the type “E” electrode for measurements in the pH region 7.5 to 11.8. The peptides were dried to constant weight DISSOCIATION CONSTANTS OF AMINOACIDS, T = 2R.0°, i n vacuo over phosphorus pentoxide a t 57”, and 50 ml. of y / 2 = 0.100 solution having a peptide concentration of approximately This paper Literature 0.01 moles in 7 / 2 = 0.1000 sodium chloride were prepared. Compound f i K ’ ~ fiK’s pK’r ~ K ’ I P K ’ ~ pK’r Twenty-ml. aliquots of these solutions were titrated with Glycine 2 . 3 8 9.76 2.350° 9.781” acid or base in a 50-ml. beaker iinniersed in a constant teni- I.Leucine 9.08 9.747b perature bath kept a t 25.00 zt 0.05”. Additions of acid 9.W or base were made from burets having :i capacity of 5 ml. L-Histidine 6.12 9.17 0.10d 9.18’1 and being graduated in intervals of 0.U1 ml. The tips of 2.10 9.07 2.17e 9 . w these burets were drawn to a fine point and were kept below 1.-Arginine 1.807f 9.01’ the surface of the solutions being titrated. Stirring \vas L-Lysine 2.113 9.18 1 0 . 7 9 2.188 8.w 10.53‘ -
+
(1) Presented at the XIIth International Congress of Pure and Applied Chemistry, New York, N. Y., September, 1951. (2) U. S. Public Health Service Postdoctorate Research Fellow
1919-1951. (3) Department of Biochemistry and Nutrition, Graduate School of Public Health, University of Pittsburgh, Pittsburgh 13, Pa. (4) E. Brand and B. F. Erlanger, THISJOURNAL, 7 8 , 3314 (1950); B. F.Erlanger and E. Brand, ibid.. 73, 3508, 4025 (1951); E.Brand, B. F. Erlanger, €1. Sachs and J. Polatnick, i b i d . , 73, 3510 (1951); E . Brand, B. F. Erlanger, J. Polatnick, H. Sachs and D. M. Kirschenbaum, ibid., 78, 4027 (1951). (5) We acknowledge our gratitude to Dr. E. Brand for making available t o us these peptides, w m r of nhirh hsd hren nyntkedlod is very rrnnll q w r i t l t i r n unl,
D-Lysine L-Citrulline -1-Benzylhydrogen Lglutamate ?-Ethyl hydrogen Lglutamate Citric acid
3 . 1 5 9.16 i n . 5 1 2.43 9.41
2.17 9.00
3.12 4.71
2.148’ 9.190 6.23 3.OSh 4.74*
6.26’
Owen THISJOURNAL, 56, 24 (1934). *Smith, Taylor and Smith, J . Biol. Chem., 122, 109 (1937). CMiyamoto Greenstein, ibid., 93, and Schmidt, ibid., 90, 165 (1931). 479 (1931). * Compilation of E. J. Cohn, Ergcb. P h siol., 33, 781 (1933). 3 Batchelder and Schmidt, J . P h y s . Zhem., 44, 893 (1940). 0 Neuberger, Biochcm. J., 30, 2085 (IQ28). Simme. 1,P h y s . Chem,, 32, 1121 (XQ2A).
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FFPECT O F OPTICAL CONFIGURATION O N nISSOCIL4TIONCONSTANTS O F PEPTIDES
TABLE I1 DISSOCIATION CONSTANTS OF PEPTIDES, T = 25.0". r / 2 = 0.100 PK'l Peptide
(-COOHI
H .Gly-Ala.OH (L ) H.Gly-Ala.OH ( D ) H.Ala-Gly.OH (L) H.AIa-Gly.OH (D)
3.17 3.16 3.17 3.20
H.Gly-Ala-Ala.OH (LL) H.Gly-Ala-Ala.OH (LD) H.Ala-Ala.OH (DD)* H.Ala-Ala.OH (LD)' H.Ala-Ala-Ala.OH ( 3 ~ ) H.Ala-Ala-Ala.OH (LLD) H.Ala-Ala-Ala.OH (LDL) H.Ala-Ala-Ala.OH ( DLL) H-Ala-Ala-Ala.OH ( 3 ~ ) H .Ala-Ala-Ala-Ala.OH ( 4 ~ ) H.Ala-Ala-Ala-Ala.OH ( LLDL) H.Ala-Ala-Ala-Ala.OH (LDLL) H .Ala-Ala-Ala-Ala.OH ( DLLL) H.Lys-Ala.OH (LL) H.Lys-Ala.OH (LD) H .Ala-Lys-Ala,OH ( 3 ~ ) H.Ala-Lys-Ala.OH (LDL) H.Ala-Lys-Ala.OH ( LLD) HaAla-Lys-Ala-Ala.OH ( 4 ~ ) H .Ala-Lys-Ala-Ala.OH (LDLL)
3.38 3.30' 3.30 3.12 3.39 3.37 3.31 3.37 3.39 3.42 3.24 3.22 3.42 3.22 3.00 3.15 3.33 3.29 3.58 3.32
PK'x a-NHz)
PK'5 :€-NH~)
Charge separation,
R , in
A.
5.26 5.26 5.26 5.27 6.05 5.97 5.64 5.44
8.23 8.24 8.18 8.19 8.10 8.17
8.14 8.30 6.22 8.03 6.20 8.05 6.14 8.13 6.20 8.06 6.22 8.06 6.72 7.94 6.49 7.93 6.47 7.99 6.72 7.99 6.31 10.70 7.62 6.09 10.63 7.74 6.59 10.30 7.65 6..83 10.36 7.97 6.78 10.49 7.84 7.46 10.58 8.01 7.14 10.37 8.01 7.73 10.35 7 . 7 5 HaAla-Lys-Ala-Ala-AlaeOH (5~) 3.53 7.41 10.29 H .Ala-Lys-Ala-Ala-Ala.OH (LDLLL) 3.30 7.85 10.05 11.01 6.71 7.53 H.Lys-Lys.OH ( L L ~ 3.01 9.92 10.89 6.53 7.53 2.85 H .Lys-Lys.OH ( LD) 11.32 7.46 9.80 10.54 7.34 H.Lys-Lys-Lys.OH ( 3 ~ ) 3.08 11.42 7.26 9.79 10.54 7.29 H.Lys-Lys-Lys.OH (LDL) 2.91 11.097.31 9.60 10.38 7.14 H.Lys-Lys-Lys.OH (LDD) 2.94 a H . L ~ s - L ~ s . O H ( Lwas L ) prepared first as the dihydrochloride by Dr. B. F. Erlanger and the autho; according to the method of R. A. Boissonas, Helv. Chim. Acta, 34, 894 (1951); N, 15.8 (theory 16.1); T\;Hz;N, 12,2 (12.1); HCl, 21.3 (21.0), neut. equiv., 116 (115); [a]% +12.8 ( c 2, 0.5 N HC!). "Identical with(LL). Identlcal with (DL). The preparation of these compounds will be described in another publication.
L-lysine, D-lysine, L-citrulline, y-benzylhydrogenL-glutamate and citric acid. The values for lysine differ markedly from these previously reported. In an effort to investigate this discrepancy, titrations were carried out on lysine solutions covering the concentration range 0.0020 to 0.0200 molar, but the results obtained fell always within the limits of experimental error (maximum spread of individual pK' values *0.03 pK' units from average). The values for lysine are presented here as new values, and so are those for L-citrulline. Citric acid was included in order to show that the method of resolving the dissociation of overlapping groups suggested by Greensteins and employed in this study gives valid results. Peptides.-The dissociation constants of the peptides studied are listed in Table 11. The notation is that of Brand and Erlanger, who introduced it in 19504 to symbolize amino acid residues. Greenstein's6 method of resolving overlapping dissociation constants was adopted, because the limited precision of the second decimal place of the PH measurements did not warrant more elaborate (6)
1, Orecaotein,
J , B i d . Chrm., $ 8 , 470 (leal).
methods of resolution such as those employed by Bates and Pinching,? nor were the necessary peptides and derivatives available in sufficiently large quantities. Results obtained compare well with those computed by the more elaborate method due to Speakmans which recently was applied in a similar study by Peek and HilL9 The titration curves of three groups of peptides, namely, dialanine, alanyllysine and trilysine, are shown in Figs. 1 , 2 and 3. The points shown in the first two figures are experimental points, while the curves drawn represent the titration curves computed from the pK' values listed in Table 11. For clarity's sake, experimental points have been omitted in Fig. 3, since approximately eighty pH measurements were made for each titration curve.
Discussion From the data in Tables I and I1 it is seen that the dissociation of the ionizable groups in amino acids and peptides containing but one optically (7) R. G . Bates and G. D. Pinching, TXIS JOURNAL, 71, 1274 (1949). ( 8 ) J. C. Speakman, J . Chcm. Soc., 866 (1940). (9) H.M, Peck and T . L. Hill. Txre JourrcM., TS, 6804 (1061).
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optically active residue gives rise to effects which appear to be somewhat T=250” different from those responsible for the changes observed with peptides ‘1 containing only two optically active residues. The dissociation constants for four trialanine peptides are essentially identical, provided that the enantiomorphous residue is in a terminal position, but placing it in the endo position (LDL) gives rise to small, but significant changes in both p K f values, in the directions observed with the dipeptides. I n the tetraalanine series, pK‘1 is lowered only when the D-residue is placed in an endo position. The change in the dissociation of the a-amino group may be attributed either to some 3 4 5 6 7 9 10 short range effects due to a D-residue RHI which influences pK’2 only when it is Fig. 1. no further than one residue away from the a-amino group, or to exTITRATION CURVES OF H,Ala-Lys-Ala.OH.HCI perimental error (pK’2 = 7.96 =t 0.03). Unfortunately, this series ‘I T= 2 5.0” could not be extended to higher alanine peptides, because of their greatly reduced solubility in aqueous media. I n amino acids the pK’s of the carboxyl and a-amino groups are markedly different from those of either fatty acids or aliphatic amines. Charge, dipole induction, and effects of resonance may be responsible for such changes in dissociation when both types of groups become part of the same molecule and are linked to a common carbon atom. An increase in the distance separating these two charged groups would be expected to bring about a change in the ease of dissociation. 3 4 5 6 7 8 9 10 II I n peptides, PKI becomes greater, PH. but bK2 becomes smaller than in the Fig. 2. amiio acids. This is as expected, beactive amino acid residue, is independent of the cause replacement of -COOH by -CONH- in an optical configuration of the asymmetric carbon a-amino acid greatly reduces the basicity of the atom. The following pK’ values may be assigned amino groups.l0 I n the alanine peptides containing two centers of asymmetry, the relative shifts in to these peptides pK’1 and pK’2 can be accounted for probably by a PK‘1 = 3.18 6 0.02 H.Gly-Ala .OH combination of all three of the above effects. In or or D ) the tri- and tetrapeptides of alanine, pK’1 seems to PK‘, = 8.21 f 0.03 H .Ala-Gly .OH be much more sensitive to a change of optical Such a behavior could not be expected if the forma- configuration than PK‘z, and it may well be that tion of the dipeptide gives rise to a compound one should consider other phenomena, such as having two asymmetric carbon atoms, as in di- restricted rotation of atoms adjacent to the peptide alanine, lysylalanine and dilysine. I n these pep- “backbone,” or a change in the property of the tides, as well as in the tripeptide glycyldialanine solvent structure surrounding the peptide linkages. which also has only two centers of asymmetry, the Such a simple picture cannot be expected to hold introduction of an enantiomorphous residue is for peptides containing an additional charged accompanied b y a decrease in pK’, and an increase group, such as the e-amino group of lysine. From in pK‘2. An exception is dilysine, where the the data in Table I1 no definite trends can be dissociation of the a-amino group apparently re(10) E. J. Cohn and J T Edsall, “Proteins, Amino Acids and Pepmains unchanged, while the e-amino group becomes tides a9 Ions and Dipolar Ions,” Reinhold Publishing Corp., N e w more acid when a D-residue replaces L-residue. York, N Y , 19.17 T t e e4pr1 1.1l1v < hapier. I\’ atid V for fidl d i i c i i z Extending the length of the peptide chain by one 5 I O i l TITRATION C U R V E S
OF H Ala-Ala.OH
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E F F E C T OF OPTICAL CONFIGURATION ON nISSOCIATION CONSTANTS O F PEPTIDES
detected. Lysylalanine resembles the other dipeptides; as to the higher lysine-containing peptides, no interpretation can be offered concerning the observed changes in pK' values. The introduction of the charged epsilon amino group undoubtedly sets the stage for a whole series of interactions among all charged groups, so that the simple picture outlined for monoamino-monocarboxy peptides cannot be applicable. Let us assume that the pK', values are affected only by the proximity of the charged a-amino group in the simple peptides, and let us further assume that in peptides containing the lysine residue this charge is replaced by an imaginary charge placed a t the locus of all positive charges. We then make use of the theory developed by Westheimer and Kirkwood,'l considering the peptide as an ellipsoid of revolution with two charges placed a t the foci. The peptide itself is considered as a cavity of low dielectric constant, and a value of 2.00 units is assumed for this internal dielectric constant, DE. The distance separating the two charges is calculated from R3 = 6V/n(Xo3 - Xo)
TITRATION CURVES OF H.Lys-Lys-Lys.OH.3HGI
T=25.0°
(2)
where R is the distance in Angstroms between the charges, V the molar volume, and XOthe equation of the ellipse, obtained from &(charge)
(hoS - XO)'/a
-
e2(7r/6 V)'/a
- 2.303BTApK
5201
PH*
(3)
where e is the electronic charge, k Boltzmann's constant and T the absolute temperature. ApK has been chosen as the change in pK', caused by the presence of the charged group, using the dissociation constants of fatty acids as reference. For the calculations P K f a t t y acid was chosen as 4.76.12 The left hand member of (3) has been tabulated by Westheimer and Kirkwood.'l For our calculations we have employed the residue partial specific volumes in the computation of V,l0 rather than those obtained from Traube's rule. R values calculated in this manner are listed in the last column of Table 11. These values are considerably smaller than those obtained from crystallographic data.13 I n aqueous solutions one would not expect these compounds to exist in the extended zigzag chain.1° The repeat (11) J. G. Kirkwood and F. H. Westheimer, J. Chcm. Phys., 6, 506 (1938); F. H. Westheimer and J. G. Kirkwood, ibid., 6,513 (1938). (12) We agree with the referee who pointed out that the effect of the peptide linkage on $K'I is being ignored by this choice, and t h a t a value of 3.6 ( p K of acetylglycine, ref. 10) would be much more realistic. We had a t one time employed this a s t h e reference value, but found that it led to impossibly large R values for many compounds. (13) R. B. Corey and J. Donohue, THIS J O U R N A L , 72, 2899 (1950).
Fig. 3.
distance of the extended form has a value of 7.27 k ,yhich would mean a charge separation of about 9.7 A. for tripeptides, 13.4 A. for tetrapeptides, and 17.0 A. for pentapeptides. The computed R values are considerable smaller, and in view of the assumptions made, should not be considered to be better than of the right order of magnitude. The experimental data lend themselves to one further observation. Examination of the PK' values listed in Tables I and I1 would lead to the conclusion that some values hitherto assigned to certain ionizable groups in proteins and amino acids might be due for revision. The decreasingly smaller increments of the dissociation constants with increasing number of amino acid residues up to five indicate that PK' for the terminal a-COOH group would be around 3.7, for the terminal aNH2 group around 7.8, and for the e-amino group around 10.6 in an average protein. Acknowledgment.-The author wishes to thank Mr. Donald M. Kirschenbaum for his valuable assistance in this investigation. PITTSBURGH 13, PA.
