hlarch 5 , 1960
ASSOCIATION OF DIVALENT CATIONSWITH ACYLATED HISTIDINEDERIVATIVES 1107
ORGANIC AND BIOLOGICAL CHEMISTRY [ CONTRIBUTIOX FROM
THE
BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY]
The Association of Divalent Cations with Acylated Histidine Derivatives1 BY
R. BRUCEMARTINAND JOHN T.
EDS.4LL
RECEIVEDAUGCST12, 1959 The association of the divalent cations copper, nickel, zinc, cobalt and cadmium with acetyl-L-histidine, P-alanyl-L-histidine (carnosine), glycyl-L-histidine and histidylhistidine has been studied by potentiometric methods. Copper( 11) and nickel(I1) ions promote the ionization of the peptide hydrogen in carnosine and histidylhistidine and both of these metals as well as zinc(I1) induce this ionization in glycylhistidine. Copper(I1) and nickel(I1) ions also cause the ionization of the hydrogen bound at the pyrrole nitrogen of the imidazole ring in glycyl- and histidyl-histidines. I n the case of nickel( I1 ) complexes of the two latter peptides, the color of the solution changes from blue to yellow in the later stages of the titration with alkali. I n the yellow products the maximum in the absorption band is found a t 450 m9, and the molar extinction coefficient is 110.
The acid dissociation of the amide hydrogen in ide or other ionizations. No attempt was made to certain glycine peptide complexes of copper(I1) and determine values of association constants for log k nickel(I1) ions has been demonstrated.* The bind- less than 1.5. The pK values of the additional ing of various divalent cations t o imidazole has also acidic ionizations from the complex were determined received extensive study. This paper is concerned in high concentrations of equimolar mixtures with a conjunction of these two lines of study, in- of amide and metal(I1) ion and evaluated as prevolving the effect on the amide linkage of the bind- viously described. The method employed gives ing of divalent metal a t the imidazole group in precise PK values only for the highest pK observed. acylated histidine derivatives. The compounds The value quoted for pKl is a maximum value, the investigated in this research are acetyl-L-histidine, error being greatest for the copper complexes. p-alanyl-L-histidine (carnosine), glycyl-L-histidine TABLE I and histidylhistidine. With regard to metal binding, the last compound is in a class with histi- FORMATION AKD IOXIZATION CONSTAXTS FOR ACYLATED dine and its derivatives which contain a free a- HISTIDINESWITH DIVALEXTMETALIoss AT 25' AXD 0.16 amino group but is included since the results obIOSICSTREXGTH tained are not unrelated to the other compounds cu Ni Zn co Cd studied. Among the systems reported here, only Acetyl-L-histidine the interaction of copper(I1) ion with carnosine has p K , = 7.08 (imidazole) been studied previously. 2.35 2.70 2 85 2.50 logk1 4.35
Experimental
The techniques have been previously described.2 The temperature was 25' and the ionic strength about 0.16 throughout the study. Acetyl-L-histidine monohydrate and carnosine were obtained from the California Corporation for Biochemical Research. Glycyl-L-histidine hydrochloride monohydrate was obtained from Mann Research Laboratories. The foregoing products were of good quality. Histidylhistidine was obtained from Mann Research Laboratories, lot S o . B 1324, and Sutritional Biochemicals Corp. Titration of either of these products with one equivalent of base or two equivalents of acid indicated t h a t approximately 80% of both preparations consisted of histidyl-histidine; they were presumably identical. Since the equivalent weight obtained by titrating with either acid or base was the same the indications were that the impurities are either water of hydration or inert salt. Drying to constant weight indicated t h a t 207" of the original material was volatile a t 110' in vacuo. Thus the sample contains 4 .O moles of water of hydration per mole of histidylhistidine. T h e optical configuration of the histidine residues in the peptide was not specified by the manufacturer and remains undetermined.
Results The results are tabulated in Table I. The successive formation constants were determined a t high ratios of amide to divalent metal ion in order to drive the equilibria toward the formation of the higher complexes and to minimize the effect of am(1) This work was supported by grants from t h e National Science Foundation (G-3230) and t h e United States Public Health Service (H-31li0). (2) R.B. Axartin, hl. Chamberlin and J. T. Edsdll. THISJ O U R N A L , 81, 49.5 (1960). References t o earlier studies are given in this paper. (3) H. Dobhie and W. 0. Kermack, Biochcm. J., 69, 246 (1955).
log kz logka log ka
3.40 2.55 1.5
2.20 1.7
2.30 2 15 1.8
1.80
1.95
L- Carnosine
p K , = 6.86, 9.40 (imidazole, a-amino) log ki 4.2 2.80 2.30 2.25 log kz 2.10 2.10 1.6 log k3 log kr
PKl PKz
1.6
5.00 5.55
2.50 1.75
2.00 1.7
7.35 8.40
8 . 50
Glycyl-L-histidine
pK, PK1 PK2 PKa
= 6.79, 8.20 (imidazole, a-amino)
4.00 4.50 9.25
6.10 6.70 9.25
6.50 7.10
7.30
Histidylhistidine
PK. = 5.40, 6.80, 7.95 (2 imidazole, 1 a-aniiiiu) 6.10 PK1 4.40 7.80 PK2 6.15 10.25 10.25 PKI
For the copper complexes of acetylhistidine additional ionizations are observed, which may be, a t least in part, amide hydrogen ionizations. However, since they occur a t pH values greater than 8 and precipitates ultimately form, it is likely that they are due to hydroxo complex formation and
R. BRUCEXARTIN AND JOHN T. EDSALL
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1 2 2 4 j 6 7 8 Moles base per mole metal ion. Fig. 1.-Titration curves of solutions of glycyl-L-histidine Jb metal ion. The numeral near t h e hydrochloride and curves indicates the molar ratio of peptide t o divalent metal ion. Arrows indicate onset of precipitation. Above p H 8 the curves for copper and nickel are similar, so t h a t the solid lines represent both ions in this region,
0
01ation.~ This effect should be investigated further. I n contrast with a previous f o r m ~ l a t i o n ,the ~ formation constants of carnosine are calculated on the assumption that the initial association of the metal ion occurs a t an imidazole nitrogen rather than a t the amino nitrogen. This is a good approximation in the case of carnosine because though the formation constants of imidazole and ammonia with metal ions are comparable,; imidazole is a much weaker base and hence will be the more likely to react in the lower p H range. I n the p H region where both the imidazole and amino groups are predominantly in the basic form, the metal will be more evenly distributed between the two sites. Little chelation is likely initially a t either site as it would involve the formation of seven-membered, or even larger, rings. No attempt was made t o evaluate the higher formation constants for copper because of the low p H a t which the first ionization from the complex occurs. The pk', value of S.50 for the equimolar cadmium complex of carnosine as compared with 9.40 in the free peptide is about what would be expected on electrostatic considerations for the value of the amino ionization with an additional positive charge at the imidazole ring. This supports the formulation in terms of initial binding at the imidazole ring. Precipitation of the zinc complex prevented the determination of pK,, and oxidation of the cobalt complex occurred before this p H region could be attained. ( 4 ) A. E. Martell, S. Chaberek, Jr., R. C . Courtney, S. Westerback and 1%. Hyytiainen, Tms J U C R S A L , 79, 3OXG (1457); R . C . Courtney, IC. L. Gustafson, S. Chaberek, J r . , and A. E . l l a r t e l l , ibid., 81, 519 (1959); R . L. Gustafson and A. E . Martell, ibid.,81, 525 (1959). (5) I
