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1 0 9 8 W. LOHMANN

Charge-Transfer Interactions of Aliphatic Amino Acids with Metal Ions

WOLFGANG LOHMANN

Institut für Strahlenschutz, GSF, Neuherberg bei München und Physikalisch-Chemisches Institut der TU München

(Z. Naturforsdi. 26 b, 1098—1101 [1971] ; eingegangen am 16. August 1971)

The effect of various non-SH-containing amino acids on an electron spin resonance spectrum as well as on an optical absorption spectrum of an aqueous copper (II) solution was investigated. The data obtained indicate that a charge-transfer complex is formed with amino acids acting as electron donors. A plot of the wavelength of the CT-band vs. the pK values of the carboxyl group exhibits a straight line with the larger pK values appearing at shorter wavelengths. Similar results were obtained when the intensity was plotted vs. the pK values of the amino group; again, higher pK values correspond to lower intensities. Thus, the method allows the determination of the pK values of prosthetic groups of amino acids. The data obtained are in good agreement with the CT-theory.

For several years there have been speculations about the possible role of charge-transfer (CT) com-plexes in different biological systems1 '2. Most of the investigations dealt with 7i — n CT-complexes; however, complexes of the n — n type are also pos-sible since many biomolecules contain suitable lone-pair electrons. Considerable progress was achieved in the case of amino aoids and proteins3; their electron donor properties seem to be well estab-lished. Studies on nucleic acids and their com-ponents (bases, nucleosides, nucleotides4) showed purine derivatives to be electron donors, too. More-over, stability constants of the complexes obtained indicated that the pyrimidine derivatives are weak donors only. In the case of uracil and its halo-genated derivatives acceptor properties cannot be excluded. This would support the theoretical pre-dictions by BRILLOUIN 5 who assumed that the argenine-thymine and guanine-cytosine interactions are possibly donor-acceptor interactions.

Most of the investigations have been done with chloranil (tetrachloroquinone), iodine, oxygen or flavines acting as acceptors. Little attention has been

Requests for reprints should be sent to Prof. Dr. W. LOH-MANN, Institut für Strahlenschutz, GSF, D-8042 Neuher-bergjMünchen, Ingolstädter Landstr. 1.

1 A. SZENT-GYÖRGYI, Bioenergetics, Academic Press, New York 1957.

2 D . D . ELEY, M . M . C. DAVIES, and R . S. SNART, Nature [London] 188. 724 [1960].

3 J. B . BIRKS and M . A . SLIFKIN, Nature [ L o n d o n ] 1 9 7 , 4 2 [1963].

4 J. C . M . TSIBRIS, D . B . MCCORMICK, and L . D . WRIGHT, Biochemistry 4, 504 [1965].

given to the involvement of metal ions, although many biomolecules are known to contain them. Their structural incorporation as well as their func-tions are still unsolved in many instances.

The observations that some metal ions are present in deoxyribonucleic acid (DNA) 6> 7 and that the addition of copper(II) ions reversibly denatures D N A 8 stimulated the interest in the interactions between the bases and metal ions. These interactions might have a marked effect on the key hydrogen bonds in biological systems. In addition, the im-portance of metal ions for some enzyme reactions and for electron transfer processes9, such as in photosynthetic units and in mitochondria, stimulated our interest to investigate the interactions between aliphatic amino acids and copper(II) ions in regard to charge-transfer complexing.

Recently we have shown that SH-containing sub-stances, such as cysteine and red. glutathione, form CT-complexes with copper (II) ions 10. In the present work we have extended these investigations to non-SH-containing amino aoids.

5 L . BRILLOUIN, i n : Horizons in Biochemistry, M . KASHA and B. PULLMAN eds., Academic Press, New York 1962, p. 295.

6 J. E . MALING, L . S . TASKOVICH, and M . S. BLOIS, Biophys . J. 3 , 7 9 [1963].

7 M . S. BLOIS, J. E . MALING, and L . S. TASKOVICH, Biophys . J, 3, 275 [1963].

8 G. L. EICHHORN, J. Amer. chem. Soc., Advances in chem. Ser., 62 ,378 [1967].

9 R. J. P. WILLIAMS, Endeavour 26, 96 [1967]. 1 0 W . LOHMANN, M .MOMENI, and P. NETTE, Strahlentherapie

134, 590 [1967].

CHARGE-TRANSFER INTERACTIONS 1 0 9 9

Materials and Methods

The amino acids used for this investigation were obtained from Mann Research Laboratories, Inc., New York, New York, and were of reagent-grade quality. They were used without further purification. Different concentrations of the amino acids were prepared by dissolving them in aqueous solutions of 0.15 mM CuCl2 (optical absorption measurements) and 150 mM CuCl2 [electron spin resonance (ESR) studies]. Doubly distilled water was used as the solvent throughout the experiments.

The optical absorption spectra were determined with a Cary 15 spectrophotometer. In each case the dif-ference spectra (mixture of amino acid and cop-per (II) ions vs. amino acid) were recorded. The ESR spectra were obtained with a Varian V4502 100-kc ESR spectrometer using a liquid sample accessory. A DPPH (diphenylpiorylhydrazil) standard (g = 2.0036) was used as a reference for marking resonance posi-tions. The first derivative of the absorption curve was recorded.

Results and Discussion

Fig. 1 shows the effect of different amino acidi on the copper(II) ESR signal used as a control. The spectra obtained were identical for all non-SH-containing aliphatic amino acids. The hf splitting occuring with increasing concentrations of amino acids strongly suggests an interaction between the two constituents.

150 mM Cu 2 ein H^O

The formation of such a charge-transfer complex is also confirmed by optical absorption studies (see Fig. 2) . As can be seen in these difference spectra, a new broad band appears with increasing concen-tration of glycine; the copper(II) ion concentration

a> o S 0.6 •8 o <0 -Q

OA

0.2

0.0

Fig. 2. Difference spectra between glycine and mixture of Cu 2 0 and glycine in an aqueous solution.

was kept constant: 0.15 MM. An increase in inten-sity and a red displacement occur with increasing glycine concentration. It should be pointed out that these complexes are rather stable since there was no change with time in the spectra.

In general, the same results were obtained with other aliphatic amino acids. However, a comparison of the CT-bands of different amino acids of the same concentration (5.0 MM) showed that the bands were located at different wavelengths and exhibited dif-ferent intensities depending on the amino acid used.

A decrease in maximum absorption frequency is, according to M u 11 i k e n's CT-theory, equivalent to a decrease in the dissociation constant of the complex. Since the functional groups of the amino acids are involved in the formation of complexes with metal ions it should be of interest to find out the existence of a possible correlation between the observed optical data and the pK values of amino acids.

In Fig. 3, the pK values of the carboxyl group are plotted vs. the wavelength of the CT-bands using a constant concentration of the various amino acids (5.0 MM). The values of glycine and serine were used for calibrating the curve. Whenever the values agreed with those ones, wich were also obtained by other methods, they were marked with a dot (•). A

0.15 mn Cu 2®in H20

1. Control - 2.50mM Glycine only

3. 0025 mM " " 4. 0.1 mM 5. 0.5 mM " -+Cu2® 6.1.0 mM

I \ 7. 5.0 mM

Ä\ „ \ 7

- * V \ W \ \ V \ W

3 V O v n v v

V i i t i 200 250 300 350

A [nml 3

1 1 0 0 W. L O H M A N N

1.7 1.8 2.0 2.2 2.4 2.6 p/r, (cooh) >-

Fig. 3. Plot of the CT-absorption band (wavelength) vs. the pK values of the carboxyl group (explanation see text) .

A characterizes the values which differ from the ones previously reported. Finally, open circles (O) represent values previously unknown.

As can be seen, most of the values are located on a straight line which can be expressed as ZcT-band = - 0.7536 pK + b with 2 nm ^ 0.05 pK units. The exceptions are the diamino, monocarboxylic acids (ornithine, lysine) and the monoamino, dioarboxy-lic acids (aspartic acid, glutamic acid). The devia-tions obtained with ornithine and lysine are probably caused by the presence of HCl. It is inter-esting to note that the other diamino, monocarboxy-lic acids, arginine and citrulline, act like mono-amino, monocarboxylic acids indicating that the amino group in <5 position does not participate, to any larger extent, in the formation of the complex.

The results obtained are in very good agreement with the CT-theory: an increase in the wavelength of the CT-band is equivalent to a decrease in the dissociation constant. Moreover, methylation causes a decrease in the ionization potential resulting in an increase in the wavelength of the CT-band and,

thus, in a decrease in the pK values. The data ob-tained confirm this prediction: the CT-band of sar-cosine, a methylglycine, is located at a considerablv higher wavelength that the one for glycine.

A plot of the intensity of the CT-band vs. the pK values of the amino group, using alanine and serine for calibration, exhibits again a straight line for most of the amino acids tested (see Fig. 4) . Again, deviations were obtained with the same amino acids mentioned in Fig. 3. The intersection of the measured intensity of ornithine and lysine with the lower dashed line and the solid line, resp., results in the pK values of the two amino groups of those two amino acids. The same applies for asparagine and glutamine using the solid line and the upper dashed line.

The values obtained by this method agree with the ones obtained by other methods with the excep-tion of leucine, isoleueine, and threonine (see Figs. 3 and 4) . Based on our results, the following pK values were obtained (the average values obtained by other methods are given in parenthesis) :

pKx: Thr 2.16 (2.088), Leu 2.195 (2.344), lieu 2.17 (2.339).

pK2: Thr 8.28 (9.1), Leu 9.39 (9.672), lieu 9.55 (9.72), Glu 10.5 (9.53).

An explanation cannot be offered for this deviation as yet. In a few cases, it has been possible to deter-mine the pK values for the first time.

An interesting result is obtained when the cop-per (II) ion concentration, at constant amino acid concentration, is varied. In this case (see Fig. 5), the wavelength of the CT-band remained unchanged (at 211 nm) ; only the intensity increased propor-tionally to the copper concentration.

A 0.6 _ Dipa Asp-NH2

\ 0.4 -

0.2-

0.0

Olu-NH2 Asp-NH2

_GIu-NH2 n' — — —

5.0 mM AA 0.15mM Cu2®

y=-0.1228x*b [0.05 OD = 0.1 pK units]

7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 pK2 (NH®)

11.5

Fig. 4. Plot of the CT-absorption band (intensity) vs. the p K values of the amino group (symbols see Fig. 3 ) .

SCHWINGUNGSSPEKTRUM YON (C1NC0)S 1 1 0 1

1.1

A 1 0

1 0.8 Qt c ns •eo.5 o <0 ^0.4

0.2

00 0.1 0.2 03 0.4 0.5 0.6 Cu 2®-conc. [mM] »•

Fig. 5. Intensity of the CT-band vs. varying concentrations of Cu 2 0 using constant concentration of glycine (0.5 mM).

From the results obtained it might be concluded that the amino acids investigated form a charge-transfer complex with copper(II) ions. These com-plexes are rather weak as indicated by the ESR

spectra. This is in agreement with PEARSON'S clas-sification according to which the two prosthetic groups (NHo, COO e ) , available for complex for-mation, are hard ligands n . With metal ions, they undergo electrostatic or d covalent interactions. This interaction is the stronger the smaller the ionization potential of the donor (amino acid). Thus, the most stable complexes have the largest absorption wavelength. As demonstrated above, this method is useful for determining pK values of amino acids. The values obtained are very ac-curate; the standard deviation is less than 2 percent.

The excellent technical assistance of Mrs. G U D R U N W I E S E is greatfully acknowledped. This work was sup-ported in part by Fraunhofer-Gesellschaft.

1 1 R . G . PEARSON, J. A m e r . d iem. Soc. 8 5 , 3 5 3 3 [ 1 9 6 3 ] .

Das Schwingungsspektrum der Trichlorcyanursäure, (ClNCO)3

The Vibrational Spectrum of Trichloroisocyanuric Acid, (C1NCO) 3

K . DEHNICKE u n d H . LEIMEISTER

Fachbereich Chemie der Philipps-Universität Marburg/Lahn

(Z. Naturforsch. 26 b, 1101—1104 [1971] ; eingegangen am 4. August 1971)

The vitrational spectrum (IR and R a m a n ) of trichloroisocyanuric acid, (CINCO) 3 is re-corded and assigned on the basis of a planar molecule with symmetry D3h .

Die Ausdehnung unserer Untersuchungen über Substitutionsreaktionen mit Verbindungen, die elek-tropositives Halogen enthalten1, auf CINCO und (CINCO) 3 2 ließen es wünschenswert erscheinen, Informationen über die Strukturen dieser Verbin-dungen zu erhalten. Während über CINCO3 und (G1NC0)2 4 bereits vollständige Schwingungsspek-tren vorliegen, ist für die trimere Form dieses Systems bislang nur das IR-Spektrum im Bereich von 4000 —650cm - 1 bekannt5. In der vorliegen-den Arbeit berichten wir über die Ausdehnung der Messung des IR-Spektrums bis 200 cm - 1 sowie übeT das bisher unbekannte R a m a n - Spektrum des (CINCO) 3 , weil nur aus der Kenntnis des gesam-

Sonderdruckanforderungen an Prof. Dr. K. DEHNICKE, Fachbereich Chemie der Philipps-Universität, D-3550 Mar-burg/Lahn, Lahnberge.

1 K. DEHNICKE, Angew. Chem. 77, 22 [1965]; 79, 253 [1967]. 2 H . LEIMEISTER U. K . DEHNICKE, J. Organometal . Chem.

31, C3 [1971].

ten Schwingungsspektrums eine sichere Entscheidung über das Vorliegen eines planaren Moleküls (D3h) oder eines nichtebenen Moleküls (C3V , Cs) getroffen werden kann.

Tab. 1 unterrichtet über Anzahl, Verteilung und Auswahlregeln der Schwingungen auf die einzel-nen Klassen für die beiden in Betracht kommenden Modelle.

Man entnimmt Tab. 1, daß sich die Erwartungs-spektren in der Zahl der IR-aktiven Schwingungen unterscheiden (10 bzw. 14). Dieses Kriterium ist jedoch von nur geringer Beweiskraft, da gelegent-lich mit zufälligen Entartungen zu rechnen ist, die eine höhere Symmetrie vortäuschen können. Hin-

3 H. H. EYSEL U. E. NACHBAUR, Z. anorg. allg. Chem. 381, 71 [1971],

4 R . E . HESTER, R . B . GIRLING U. W . GOTTARDI, Spectro-chim. Acta [London] 26 A, 2363 [1970].

5 R . C. PETTERSON, U . GRZESKOWIAK U. L . H . JULES, J. org. Chemistry25, 1595 [I960].