Interaction of the vanadyl (IV) cation with carnosine and related ligands

The interaction of the vanadyl (IV) (VO2+) cation with carnosine (the dipeptide β-alanyl-histidine) has been investigated by electron absorption spectroscopy at high ligand-to-metal ratios and at different pH values. The results show that in the
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  ~Zopyright 1996 by Humana Press Inc. All rights of any nature whatsoever reserved, 0163-4984/96/5501-2-0079 7.25 Interaction of the Vanadyl IV) Cation with Carnosine and Related Ligands EVEUNA G. FERRER, PATRICIA A. M. WLI~S, AND ENR1QUE J BARAN Quimica lnorganica QUINOR), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900), Argentina Received June 21, 1995; Accepted November 20, 1995 BSTR CT The interaction of the vanadyl IV) VO2+) cation with carnosine the dipeptide [3-alanyl-histidine) has been investigated by electron absorption spectroscopy at high ligand-to-metal ratios and at different pH values. The results show that in the range 6.0-8.5, the cation inter- acts with the imidazole group of four different carnosine molecules and points to the presence of an axially coordinated water molecule. These suppositions were confirmed by the behavior of the VO2+/ imidazole system, which was investigated under similar experimental conditions, and supported by previous ENDOR electron-nuclear double resonance) results. The study was complemented with addi- tional measurements using the glycylglycine, glycylglycine/imidazole, and histidine systems as ligands. Index Entries Vanadyl IV); carnosine; electronic spectra. INTRODUCTION Carnosine ~-alanyl-L-histidine; Fig. 1) is found in skeletal muscles and in some other tissues of all vertebrates. Although its function is unknown, it has been established that carnosine is a substrate of the enzyme carnosinase. This enzyme is activated and/or stabilized by dif- ferent metal cations and the hydrolysis of carnosine has been extensively investigated 1-3). The interaction of metallic cations with carnosine itself has also acquired certain interest 4-8). Recently, the interaction of carnosine with vanadate has been inves- tigated 9). In this paper, we present results of the interaction of this *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research 9 Vol. 55 1996  80 Ferrer, Williams, and Baran COOH 0 CH2-CH-NH-C- CH2 H N,,, N NH2 Fig. 1. Structure of Carnosine. dipeptide with VO 2 , the other relevant vanadium species in biological systems 10-14). In order to complement the obtained information and to support some of the proposals, measurements with other related ligand systems imidazole, glycylglycine, glycylglycine/imidazole, histidine) were also undertaken. Since current information concerning VO2+/peptide interactions is scarce, the present results are of great interest in relation to the bio- inorganic chemistry of vanadium, because they allow a deeper insight into possible relevant interactions of vanadium species with simple biomolecules. M TERI L ND METHODS VOSO4. 5H20) from Merck Darmstadt, Germany), carnosine from Sigma St. Louis, MO), and imidazole and glycylglycine from Fluka Buchs, Switzerland) were used as supplied. All the employed solutions were freshly prepared and used under anaerobic conditions, in order to prevent oxidation phenomena. The VO 2+ concentration was always around 5 x 10-3M. Electronic absorption spectra were measured with a Hewlett- Packard-8452 diode-array spectrometer, using 1 cm quartz cells. RESULTS ND DISCUSSION VO2+/Carnosine and VO2+/lmidazole Interactions All the measurements were undertaken with a great excess of carno- sine, in order to avoid possible hydrolysis of the cation. With a carnosine: vanadyl IV) molar ratio of 100:1 and at pH = 7.0, the generated solutions present a violet coloration with absorption bands at 752 nm ~ = 35.8M -1. cm-1), 596 nm ~ = 17.6M -1. cm 1) and 530 nm ~ = 16.8M-1. cm-1), as Fig. 2A shows. In aqueous solutions the [VO H20)5] 2+ complex presents, as known, two defined bands at 760 nm ~ = 16.0M-1. cm-1) and 625 nm Biological Trace Element Research Vol. 55 1996  VO 2+ Cation Interaction with Carnosine 81 O ~176 L ~176176 ~176176176176 5 I i lO 8 nto] Fig. 2. Electron absorption spectra of aqueous VO2+/carnosine solutions molar ratio 1:100) at pH 7.0 A) and pH 4.0 B). ~ = 7.5M -1. cm -1) 15). The fact that, in the present case, a clear split- ting of the higher-energy band is observed, suggests a symmetry reduc- tion around the metal center. On the other hand, the band positions of the carnosine complex point to a coordination that mainly involves nitrogen-donors. An earlier ENDOR investigation of this system also suggested a vanadyl IV) coor- dination through imidazole N-atoms of four different carnosine mole- cules 16). In the case of the Cu II)/carnosine system, at pH >5 and high carnosine:copper relations, the coordination also involves the imidazole N-atoms 6). These results are especially interesting for the VO2+ complex, because of the known preference of the VO2+ cation for oxygen donors 17) and because, at the investigated pH value, not only the imidazole N- atom, but also the carboxylate group of carnosine pK - 2.6 [6]), were available for coordination. Notwithstanding, it should be remarked that imidazole apparently possesses a better G-donor capacity toward VO 2+ than other nitrogen-containing ligands cf for example, Ref. 18). In order to confirm the supposed VO2+/N interactions, we have also investigated the electronic spectra of the VO2+/imidazole system. Measuring a solution with an imidazole:VO 2+ molar relation of 100:1 at pH 7.0, practically the same spectrum as that with carnosine is obtained, with bands at 756 nm ~ = 44.0M -1. cm-1), 590 nm ~ =]), and 532 nm ~ = 23.0M -1. cm-1). The recently reported [VO 1-vinylimidazole)2C1]C1 complex, in which VO 2+ is bound to four imidazole groups in equatorial positions, and one axial C1- ion, also shows similar spectral characteristics, with two absorption bands located at 769 nm ~ = 44M -1. cm -1) and 556 nm r = 12M -1. cm-]) 19). Finally, VO2+/carnosine spectra were investigated at different pH values, using either 0.1N HC1 or 0.1N NaOH to achieve the desired value. The measurements show that in the pH range between 6.0-8.5, the spectrum of Fig.2A remains intact. With increasing pH values, a shift to Biological Trace Element Research Vol. 55 1996  82 Ferrer, Williams, and Baran lower energies, together with a small intensity diminution, is observed for the absorption band at 752 nm, but the doublet remains unchanged in position, and its intensity becomes somewhat enhanced. At pH 11.0, the spectrum changes dramatically and a broad band centered at ca. 805 nm ~ = 22M 1. cm-1) together with a better defined absorption at 586 nm r = 33.5M-1. cm-1), were observed. Starting at pH 6.0 and stepwise lowering the pH value, generates a shift of the spectral bands to lower energies, up to pH 4.0, whereas the doublet structure gradually disappears. In the pH range between 4.0-2.0, the absorption bands are found at 772 nm ~ = 24.7M -1. cm -1) and 602 nm r = 12.8M -1. cm -1) cf Fig. 2B). As in this low pH range, the imi- dazole group of carnosine is protonated pK - 6.6 [6]), only the carboxy- late group is available for coordination. This clearly justifies the band displacement to lower energies. VO2+/Glycylglycine and VO2+/Glycylglycine/Imidazole Interactions The glycylglycine/imidazole system appears to be a good model for mimicing metal/carnosine interactions, with a set of two independent ligands 8,20,21). We have repeated the experiments described above, using glygly Fig. 3) alone, as well as different glygly/imidazole mix- tures. The results are summarized in Table 1 and compared with those obtained with carnosine. It is evident that with glygly alone, at pH 5.0, the interaction occurs in a way similar to carnosine with the same pH value, i.e., predominantly through the carboxylate group, or eventually through carboxylate and the peptidic carbonyl. The same behavior is true for the different glygly/ imidazole mixtures at this pH. At pH 7.0, the involvement of N-donors in bonding becomes apparent, as the two spectral bands are displaced to higher energies. Different structural possibilities for simultaneous bond- ing of the VO 2+ center, through oxygen and nitrogen donors from glygly, has recently been analyzed by Costa Pessoa et al. 22). Successive additions of imidazole show that, at the most acidic pH values, the effect of this ligand is, as expected, negligible, and, at pH 5.0, the spectra are practically the same as those recorded with glygly alone and with carnosine. Notwithstanding, at pH 7.0, important band displacements occur, and spectra which are very similar to those mea- sured with carnosine are observed, suggesting again the predominance of N-donors. It is probable that at this pH value coordination takes place only through imidazole moieties, although the participation of some donor atom of glygly cannot totally be excluded, because of the different structure of the higher energy-band in the present case, an aspect commented upon in the next paragraph. A detailed comparison of the spectra obtained with carnosine, glygly, and glygly/imidazole shows that, in the first case, the higher Biological Trace Element Research Vol. 55 1996  V02+ Cation Interaction with Carnosine 83 Fig. 3. H2N CH2 C N CH2 COOH H Structure of glycylglycine glygly). Table 1 Electronic Spectra for the Systems VO2+/Carnosine, VO2+/Imidazole, VO2+/Glygly, and VO2+/Glygly/Imidazole System Molar ratios pH Band position nm) VO2+:carnosine I:I00 7.0 752 596/530 VO2+:carnosine i:i00 4.0 772 602 VO2+:carnosine i:i00 ii.0 ~805 586 VO2+:imidazole i:i00 7.0 756 590/532 VO2+:glygly i:i00 7.0 740 580 sh) 504 VO2+:glygly i:i00 5.0 774 596 564 sh) VO2+:glygly:imidaz~ i:i00:I 7.0 740 582 sh) 500 VO2+:glygly:imidaz. i:i00:i 5.0 770 582 558 sh) VO2+:glygly:imidaz. 1:100:30 7.0 758 582 sh) 524 VO2+:glygly:imidaz. 1:100:30 5.0 770 582 558 sh) VO2+:glygly:imidaz.l:lO0:lO0 7.0 758 582 sh) 524 sh shoulder. energy band presents a doublet structure at pH 70, which is absent in the spectra measured at acidic pH values cf Fig. 2). The same behavior was observed, as commented above, for the system VO2+/imidazole. In the remaining cases, the doublet structure changes in relative intensity at the different measured pH values. At pH 5.0, the doublet is conformed by a main band and a weak shoulder located at higher energies, whereas at pH 7.0 an inversion of the relative intensities is observed, i.e., the main band lies at higher energies than the shoulder cf Table 1). VO2 +/L-Histidine Interactions The characteristics of this interaction are of great importance for a better understanding of the biochemistry of vanadium, because this lig- and plays a key role in the generation and stabilization of numerous metal/protein complexes 4,14,23). Recently, Costa Pessoa et al. have thoroughly investigated the VO2+/L-histidine system by a combination of potentiometric and spectroscopic techniques 24). Therefore, we have Biological Trace Element Research Vol. 55 1996
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