A spectrophotometric study of the VO 2+ -glutathione interactions

The interaction of the vanadyl (IV) cation with reduced glutathione (GSH) has been investigated by electronic absorption spectroscopy, at different metal-to-ligand ratios and pH values. The interaction depends strongly on the initial VO2+/GSH ratio.
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   9 1991 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/91/3002-0175 $02.00 A Spectrophotometric Study of the VO 2 -Glutathione Interactions EVELINA G. FERRER, PATRICta A. M. WILLIAMS, AND ENRIQUE J. BARAN Departamento de Qufmfca, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900), Argentina Received September 12, 1990; Accepted October 10, 1990 ABSTRACT The interaction of the vanadyl (IV) cation with reduced gluta- thione (GSH) has been investigated by electronic absorption spectro- scopy, at different metal-to-ligand ratios and pH values. The interac- tion depends strongly on the initial VO2+/GSH ratio. Starting with a tenfold GSH excess, coordination takes place through the two carbox- ylate groups of the ligand, generating (at pH = 7) a blue 1:2 VO2+/GSH complex; this stoichiometry could be confirmed by photo- metric titration experiments. Higher GSH concentrations produce a violet complex, which can also be obtained by addition of GSH to the blue species. Some measurements with the three component amino acids of GSH, as well as results obtained from the VO~/GSH system, allowed a wider insight into the characteristics of this violet complex, in which the cation interacts with S and N atoms of the peptide. Index Entries: Vanadyl(IV)-reduced glutathione complexes; ef- fect of the metal-to-ligand ratios; vanadyl(IV) interactions with gly- cine, L-cysteine, and L-glutamic acid; vanadate(V)-reduced-gluta- thione interactions. INTRODUCTION Glutathione GSH), the tripeptide y-L-glutamyl-L-cysteinyl-glycine, has generally been regarded as the most abundant low-mol-wt thiol in a *Author to whom all correspondence and reprint requests should be addressed. iological Trace Element Research 75 Vol. 30 1991  176 Ferrer, Williams, and aran variety of cell types 1-4). The higher concentrations of glutathione, compared to other thiols in eukaryotic cells, suggests a role for this molecule related to the metabolism of oxygen, and a number of different enzymes utilizing glutathione can be related to biotransformations of products of oxidative metabolism. Oxygen-containing products, such as H202 and organic hydroperoxides, may be reduced by the action of glutathione peroxidase, which utilizes glutathione as a reductant. Some disulfides are also reduced by glutathione in the presence of thioltrans- ferase. On the other hand, a variety of electrophilic organic compounds are metabolized to stable S-conjugates of glutathione that are precursors of excretion products 3,4). Different studies have shown that glutathione may also play an important role in relation to the biochemistry of vanadium cf, for exam- ple, refs. 5,6). In fact, in red cells, vanadate V) is reduced almost quan- titatively to VO 2+ by glutathione, which can also act as a ligand for the generated vanadyl IV) cation 7,8). Although a great number of solution studies on metal complexes of glutathione have been published 2), the information concerning vana- dyl IV) complexes remains fragmentary. We have therefore undertaken a spectrophotometric study of the VO2+-glutathione system under differ- ent experimental conditions, in order to obtain a wider insight into the possible interaction modes of these species. Some aspects of the interac- tion of the VO 2+ cation with the single amino acid components of GSH were also analyzed. M TERI L ND METHODS Freshly prepared solutions of VOSO4 5H20 Merck) and reduced glutathione Sigma) were used under anaerobic conditions, in order to prevent oxidation phenomena. Glycine was purchased from BDH, and L-glutamic acid, L-cysteine, and NaVO3 were obtained from Merck. Electronic-absorption spectra were measured with a Shimadzu UV- 300 instrument and/or with a Hewlett-Packard 8452 A-Diode-Array spec- trophotometer, using 1-cm quartz cells. RESULTS ND DISCUSSION Glutathione Fig. 1) presents eight potential binding sites in its reactions with metal ions 2): two carboxylic acid groups, an amino group, a sulfhydryl group, and two peptide linkages. In order to establish which of these sites can interact with the VO 2+ cation, we have conducted experiments with different metal-to-ligand ratios. Biological Trace Element Research Vol. 30 1991  VO 2-Glutathione Interactions OOC 0 0 \ CH-CH2- CH2-C- NH-CH-C- NH-CH2-C O O- / I H3N CH2 SH Fig. 1. Glutathione GSH). 177 1. VO 2 § Ratio from 1/1 to 1/10 Oxygen-free solutions 10-2M in GSH and 10-3M in VO 2+) were prepared and the pH values were adjusted either with NaOH or with HC1. The following shifts of the absorption bands of the VO 2+ cation could be observed, working at pH = 7: from 770 and 625 nm in the aquocomplex 9) to 800 nm e = 18 L/M" cm) and 560 nm e = 18.5 L/M. cm) in the GSH complex, as shown in Fig. 2. The crystal field parameter 10 Dq can be directly obtained from the higher energy band, assignable to the b 2 ~ b 1 transition in the well- known Ballhausen and Gray schema 9). This value changes from 16,000 to 17,860/cm after GSH complex formation. The observed shifts are in agreement with those expected after vanadyl IV) coordination thorough carboxylate groups (9-12). The stoichiometry of the formed blue complex was determined by spectrophotometric titration (13), monitoring absorbance changes as a function of the metal-to-ligand ratio at constant wavelength 800 nm). One of such titrations is presented in Fig. 3. The results point clearly to the formation of a 1/2 VO2+/GSH complex. The same stoichiometry was earlier established by a NMR study, which has also shown coordination through the carboxylic groups working under similar conditions (14). On the other hand, our experiments have shown that at pH = 7, the vanadyl/GSH solutions remain stable up to a VO2+/GSH ratio of 1/1.5. A further diminution of the GSH concentration or a subsequent addition of VO 2+) induces the formation of a precipitate, probably VO OH)2. 2. VO 2 /GSH Ratio [Tom 1/100 to 1/4 Working in the same way as in Section 1, but with a 100-fold excess of GSH relative to VO 2+, a purple color is observed, associated with a new change in the electronic spectrum Fig. 2), which at pH = 7 shows bands at 748 nm e = 26.1 L/M 9 cm) and 562 nm e = 21.7 L/M " cm), giving 10 Dq = 17,800/cm. The position of these bands is strongly pH-dependent, as can be seen from Table 1. GSH coordination to VO 2+ begins at a pH value Biological Trace Element Research Vol. 30 1991   78 Ferrer Williams and Baran 1.4 1.2 1.0 0.8 0 Q o Q <0.6 0.4- 0.2- \ z 0 /f ~. \ / \ / \ / \ / \ / \ \ / \ \ ,,, -\ \ / x\ / \ \\ / \ I \ /I X I \ \ I /I .\ ~',,. \ I \. / ~ 500 600 700 800 [nm] Fig. 2. Electronic spectra of an aqueous 5 x 10-2M VO 2 solu- tion --); a VO2 /GSH 4.2 x 10-2M and 0.25 M, respectively) solution ...... ); and a VO2+/GSH 5.21 x 10-2M and 5.21 M, respectively) solution .... ). between 3 and 4. Table 1 also shows the position of the absorption maxima when the metal-to-ligand ratio changes between 1/100 and 1/4. The dependence of the coordination mode from the initial concentra- tion ratio is remarkable. The violet color corresponding to the initial complex is maintained, though the b 2 ---> e transition is shifted to lower energies, but never reaches the value of 800 nm, characteristic of the blue solution obtained starting with lower VO2+/GSH ratios. In order to obtain a wider insight into this aspect we have investi- gated the effect produced by the addition of a ligand excess to these blue solutions, working with metal-to-ligand ratios between 1/10 and 1/50 at pH = 7. The results, presented in Table 2, show that the addition of GSH produces, also under these experimental conditions, the violet complex. This evidence, together with the fact that the blue species cannot be obtained, even if the VO2+/GSH ratio is reduced from 1/100 to 1/4 cf Table 1), points to a higher stability of the violet complex, which, once formed, remains stable and cannot be transformed. Biological Trace Element Research Vo]. 30, 1991  VO 2--Glutathione nteractions 179 0.5- 0.4- l_d r zO.3- .< f13 13c o ~ 0.2-- 03 .< 0.1- 0 i 2 3 4 5 6 7 [GSHI/[VO z ] Fig. 3. Spectrophotometric titration of VO 2 + with GSH 0.05M) at pH = 7, under N2, and at X = 800 nm. Table 1 Electronic Spectral Data of Solutions Containing Different VO2+/GSH Ratios VO2+: GSH pH b 2 -~ blb b 2 ~ E b ] : ]00 2 630 776 3 - 4 600 776 5 585 770 6 - 7 562 762 7 562 748 ] : 20 7 562 766 ] : 9 7 562 766 I : 4 7 562 778 9 Atmosphere, N2; [GSH] = 0.21 M. Values in nm. bBands assigned according to the scheme of Ballhausen and Gray (9). Biological Trace Element Research VoL 30, 1991
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