The interaction of the vanadyl (IV) cation with chondroitin sulfate A

The interaction of VO2+ with the muchopolysaccharide chondroitin sulfate A (CSA) has been investigated by electron absorption spectroscopy and infrared measurements in aqueous solutions at different pH-values and ligand to metal ratios up to 6:1. The generation of a VO(CSA)2 species could be demonstrated. Coordination of the oxocation through the carboxylate group and the glycosidic oxygen of thed-glucuronate moieties is suggested. Infrared spectra of some poorly characterized solid VO/CSA complexes point to the same bonding characteristics. Preliminary results obtained at higher ligand to metal ratios suggest a different coordination behavior.


INTRODUCTION
N u m e r o u s evidence shows that v a n a d i u m m a y be considered an essential trace element for both plants and animals (for recent reviews cf.(1)(2)(3)).In relation to its absorption and metabolism in higher species, including m a m m a l s , its skeletal retention is of particular interest.
As s h o w n through by animal experiments, very little of the absorbed v a n a d i u m is retained in the b o d y and, apparentl)~ bone is a major sink for the retained element (4,5).Participation of v a n a d i u m in the first stages of calcification processes has also been suggested (6).
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In order to obtain an insight into the structural consequences of the incorporation of vanadium in the inorganic phase of bone, we have investigated the incorporation of VO 3-in calcium hydroxylapatite, Calo (PO4)6(OH)2 (7), a material that is considered a good "model" for such studies.This investigation shows that the incorporation of low to moderate quantities of vanadium has no important effects on the apatitic lab tice neither from the macroscopic (cell dimensions and distortive structural effects), nor from the microscopic (strength of the P-O and O-H bonds) point of view (7).
Most recently, we have also investigated the effect of the VO2+ cation on this apatite and found that it is not incorporated into the lattice but is strongly adsorbed on its surface (8).
As a logical continuation of these studies, and in order to obtain wider information on the interaction of vanadium species with hard tissues and related materials, we .havenow investigated the interaction of VO 2+ with chondroitin sulfate A, a well known acid muchopolysaccharide present in connective tissues and other mineralized systems (9,10).
Acid proteins and glycoproteins, phosphorylated amino acids and acidic sulfated polysaccharides have been found in the mineralized tissues of many organisms (10).Their widespread distribution among organisms from different kingdoms suggests a key role for them in the mineralization processess (11).
This investigation is also interes~,ing from another point of view, because most of the known anionic polysaccharides could potentially adsorb metalic cations and this type of adsorption has not been studied systematically to date (12).

MATERIAL AND METHODS
The sodium salt of chondroitin sulfate A (approx 70%, balanced with chondroitin sulfate C) from Sigma (St. Louis, MO) VOSO4 5H20 from Merck (Darmstadt, Germany), and VOC12 (50% solution) from Carlo Erba (Milano, Italy) were used as supplied.
Electronic absorption spectra were obtained with a Hewlett-Packard 8452 diode-array spectrophotometer, using 1 cm quartz cells.A WTW pH-530 pH-meter, equipped with standard glass electrodes, was used for the determination of the pH values of the investigated solutions.
The infrared spectra were recorded with a Perkin-Elmer (Nornmik, CT) 580 B spectrophotometer.Solutions were measured as thin films between AgC1 plates and solids were dispersed in KBr powder and pressed to very thin disks.In the case of solution studies, differencespectra (solution spectrum-solvent (H20) spectrum) were obtained and analyzed.
For all the experiments, freshly prepared solutions were used and all the measurements were performed under a N2-atmosphere, in order to prevent oxidation phenomena.

Electronic Spectra
Chondroitin sulfate A (CSA, Fig. 1) is a muchoplysaccharide containing alternate units of D-glucuronic acid and N-acetyl D-galactosamine.The two chondroitin sulfates, A and C, differ in the position of the sulfate group on the N-acetyl galactosamine residue and in the length of the chain (10).The array of regularly repeating carboxyl and sulfate groups generates a high negative charge on the polymer.
In a first series of experiments the electronic absorption spectra of 1:1 VO2+: CSA solutions at different pH values were investigated.
Up to pH 4.0 no interactions were observed.The spectrum remains identical to that of [VO(H20)5] 2+ (13).In the pH range 4--5 a shift of the two electronic transitions from 760 to 784 and from 625 to 610 nm is observed.These displacements clearly suggest a change of the coordination sphere around the metal center, but with persistence of oxygen donors.This statement is additionally supported by the still greater band shifts to 820 and 548 nm, observed at pH 6.0.A further increase above this pH value produces the hydrolysis of the VO 2+ cation.
Figure 2 shows electronic spectra recorded at pH values of 4.5 and 6.0, compared with the spectrum of [VO(H20)5] 2+, obtained at pH 3.5.The behavior of the system at other ligand to metal ratios, up to 6:1, is totally similar as that observed in the 1:1 system at different pH values.
In order to establish the stoichiometry of the VO2+/CSA complex in solution, a spectrophotometric titration (14) was undertaken.A series of solutions with ligand to metal ratios, r, ranging from 6 to 0.5 were prepared and their pH value fixed at 5.0.Absorbance measurements were performed at 794 nm.The plot of absorbance against r values is shown in Fig. 3.The inflection point suggests the formation of a VO(CSA)2 complex.

Infrared Spectra
In order to explore this system in more detail, an analysis of solution infrared spectra was performed.
The infrared spectra of 0.1M solutions of the free CSA were measured at three different pH values, namely 1.5, 5.0, and 10.0, and compared with the spectrum of solid CSA.Finally, the spectrum of a 2:1 CSA:VO 2+ solution, at pH 5.0 was also measured.In all the cases the spectral range between 1700-800 cm-1 was analyzed in detail.
Table 1 shows the most characteristic bands located in the mentioned range, for all the measured systems.Some of them have been assigned on the basis of some general literature references (15,16).Typical spectral patterns, useful for the structural analysis are depicted in Figs. 4 and 5. Comparing the data of Table 1 and the spectra of Fig. 4, it could be clearly observed that the Vas(SO4) band practically does not change when one moves from free CSA to the VO/CSA complex, suggesting no interaction of the cation with the sulfate groups.On the other hand, in the region of the ring modes, although the two components of the main band do not change appreciably, their intensity is strongly reduced in compari- son with that of the sulfate-band and the shoulder, located at 1129 cm -1 in free CSA is clearly displaced to 1150 cm-1 and generates a new and well-defined band.These changes suggest the participation of the C-O-C sugar units in metal-bonding.

VO2 § Interactions
In the region of the Vs (COO-) stretching vibration, the doublet at 1417/1377 cm-1 is strongly pH-dependent as can be seen from Fig. 5.In the 2:1 CSA/VO solution important changes can also be observed.Only in this case, both components of the doublet present the same intensity.This behavior allows us to suggest the participation of the carboxylate group of the glucuronic acid moieties in bonding.
Unfortunately, no effect were observed in the 1645 cm-1 band, probably because of the overlap of the Vas (COO-) mode with the strong and broad amide I band of the HN -C = O group, which frustrates the observation of eventual small band shifts and/or intensity changes of the carboxylate stretching.
The typical V = O stretching mode of the vanadyl(IV) cation could not be identified.It is surely overlapped by one of the components of the block of bands located between 1100-900 cm-1.
From the analysis of the infrared spectra it becomes evident that bonding of the VO 2+ cation occurs through the carboxyl group and the sugar oxygen atom of the glucuronic acid moieties, as depicted in Fig. 6, confirming the conclusions reached from the electronic spectra, which also suggested an oxygen environment of the metal center.
In the cases of Ca(II) (17) and Yb(III) (18), the interactions with CSA apparently takes place mainly through the carboxylate group.In the related hyaluronate polymer, constituted by D-glucuronate and N-acetyl-D-glucosamine residues, a copper(II) complex of stoichiometry CuL2 has
been reported (19,20).Its ESR and NMR data also indicate coordination through the carboxylate group and the glycosidic oxygen (21) and exclude the participation of the N-acetyl nitrogen in bonding (22).
In a more recent study of the interaction of Cu(II) with CSA, interation through the carboxylate group was also demonstrated, whereas the nitrogen atom of the N-acetyl group appeared not to be involved in bonding (23).
On the other hand, in all similar systems formerly investigated, it was also found that the sulfate groups were not involved in bonding.They participate only in electrostatic interactions with the cations (18,23).
The comparison with these previous investigations show, that VO 2+ behaves in a similar way as other metalic cations in its interaction with CSA.

Attempts to Isolate a Solid CSA/VO 2 § Complex
Although it seems difficult to obtain solid metalic complexes with ligands of the CSA-type, we have made a number of experiences in this direction.
Usually, we mixed CSA with VOC12 in a 2:1 ratio (for example, 1.0 mmol CSA with 0.5 mmol VOC12, in 10 mL of water) and adjusted stepwise the pH-value to 5.0-5.5 with diluted NaOH solution, working in a N2-atmosphere.The solvent was evaporated under high vacuum, or precipitation of solids was attempted by addition of different organic solvents.In most cases, green-colored oily products or finely powdered solid residues were collected.Their chemical analysis gave variable and poorly reproducible results.
But, despite the fact that their stoichiometries are not well defined, the infrared spectra of these solids also suggest the same type of interactions as those postulated from the solution studies.The spectra of the green powders are not very different from that of the free CSA but, interestingly, important intensity changes are observed in the 1415/1371 cm -1 doublet, suggesting again involvement of the carboxylate group in bonding.On the other hand, the m e d i u m intensity band located at 1130 cm -1 in CSA is shifted to 1125cm -1, whereas the other three components of the infrared band multiplet also show slight intensity changes, pointing to a participation of the sugar moiety in bonding.The V = 0 stretching mode could also not be identified with certainty in the spectra of these solids.

Preliminary Results at Higher CSA/V02 § Ratios
At higher ligand to metal ratios, in the range 10:1 to 100:1, other interesting aspects were detected, from electronic absorption spectra.
At pH 4.0 the behavior is similar to that described above, i. e., two bands located at 780 and 614 nm were found.But, these bands are shifted to higher energies at pH 5.0 (760 and 590 nm, respectively) suggesting the participation of the N atoms of the N-acetyl groups.Although the infrared spectra are more difficult to interpret in these cases, they show, interesting135 that the pyranose ring vibrations remain practically unaffected.
The commented observations suggest that at very high ligand to metal ratios the participation of the N-acetyl groups in bonding, cannot be excluded.This result is very interesting because no metal/amide interactions has so far been reported in chondroitin sulfate complexes (cf.ref. 24).Therefore, the system CSA/VO2+--at higher ligand to metal ratios--should be investigated in more detail, using additional spectroscopic methods in order to clarify definitively the involvement of Ndonors in bonding.The results of such a study would also be of interest in relation with the binding of VO 2+ to collagen, a system in which vanadyl-nitrogen interactions has been clearly established (25).

Fig. 5 .
Fig. 5. Difference infrared spectra of free CSA solutions at two different pH values and of the CSA/VO 2+ complex at pH 5.0 in the region of the Vs (COO-) stretching mode.