Po/yhedm Vol. 7, No. 14, pp.

124S1262, Printed in Great Britain

1988 0

0277-5387/88 $3.00+.00 1988 Pcrgamon Press plc

OXOVANADIUM(IV) AND AMINO ACIDS-I. THE SYSTEM L-ALANINE + V02+ ; A POTENTIOMETRIC AND SPECTROSCOPIC STUDY
J.

COSTA PESSOA

and L. F. VILAS

BOAS

Centro de Quimica Estrutural, Complexo Interdisciplinar, 1096 Lisbon, Portugal

Instituto Superior Tecnico,

R. D. GILLARD* Department of Chemistry, University

and R. J. LANCASHIRE?
College, P.O. Box 78, CardifTCFl

IXL, Wales, U.K.

(Received 25 January 1988 ; accepted 9 February 1988)

Abstract-The equilibria

in aqueous solution in the system L-alanine+VO*+ have been studied by a combination of pH-potentiometric and spectroscopic methods (EPR, visible absorption and circular dichroism) in the pH range 1.5-13. For pH > 4, high ligand to metal ratios were used. The results of the various methods are made self-consistent, then rationalized assuming an equilibrium model including the species MAH, MA, MAH_2, MA2H2, MA2H, MA2, MA2H_ I, M2A2H_ 2, M2A2H_3 (where HA denotes L-alanine) and several hydrolysis products ; their formation constants and individual electronic spectra (isotropic and circular dichroism) are given. The isomerism arising from the combination of the lop-sided oxovanadium ion with the asymmetric carbon ligand is analysed.

The V02+ ion is a useful paramagnetic probe of protein structure ; it would be useful to know the nature of its amino acid complexes in solution and the preference exhibited by V02+ for various potential chelating combinations with those a-amino acids like serine having additional coordinating * Author to whom correspondence should be addressed. groups. SNOW at University of the West Indies, Kingston 7, Although knowledge of such complex equilibria Jamaica. of vo2+ in the presence of amino acids is relevant $ The occurrence of vanadium in mushrooms (50 specin understanding its possible interaction with likely ies) was surveyed by H, Meisch, J. A. Schmitt and W. biological ligands, few reliable data exist. The equiRienle, 2. Naturforsch. 1978, 33c, 1, who found backlibria in aqueous solutions containing V02+ and ground levels in all but A. muscaria. The hazardous nature of vanadium, chiefly vanadate, for marine life in amino acids have been reviewed recently. I8 Several general is also documented (see NERC report 1977/8, experimental methods have been used and the isoHMSO, London (1978). lation of solids has also been claimed. 0 When solids of known structure can be isolated from This first part introduces our study and gives solutions at labile equilibrium between a metal ion and detailed results for L-alanine+VO*+ in the pH an optically active ligand, a comparison of their CD range 2-12. The development of the series will then spectra (as discs or mulls) with those of their solutions be : (2) L-serine and L-threonine : (3) L-cysteine and can be informative, as found with vanadyl(+)tartrato D-penicillamine ; (4) L-aspartic and L-glutamic species or with copper(I1) amino acid complexes (R. D. Gillard and S. H. Laurie, J. Chem. Sot., Chem. Commun. acids. Some work has been presented in a pre1969, 489; J. Chem. Sot. 1970, 59). We have not yet liminary form.224 In pH-potentiometric investigations of solutions isolated pure crystalline compounds from any aqueous oxovanadium(IV)-amino acid system. containing V02+ + N-donor ligands, relatively high The biochemistry of vanadium has attracted increasing interest, Mparticularly in studies related to its accumulation in certain tunicates7- and in the mushroom Amanita muscaria.~~7
1245

1246

J. COSTA

PESSOA

et al.

pH is necessary to achieve an appropriate free ligand concentration for complex formation. At pH > 4, however, hydrolysis of the V02+ ion takes place and oxidation to V(V) may also be significant unless careful deoxygenation is carried out. Another difficulty is that the nature (and formation constants) of the hydrolysis products of VO+ in the pH range 5-12 is not well understood. Tomiyasu and Gordon2 studied the system glycine + V02+ in the pH range 2-7 mainly by spectrophotometric measurements. They used large ratios of glycine to VO*+ to suppress metal precipitation and explained their results assuming that the system is characterized by model I in Table 1. At pH 3 7, Tomiyasu and Gordon report that, even with a glycine: vanadium ratio of 100, the absorbance of the solution decreases and the system is markedly less stable with respect to hydrolysis. Fabian and Nagypa126 studied the same system using a very high excess of glycine and characterized it assuming model II in Table 1. This is certainly a better approximation to the real situation existing in VO*+ + glycine solutions than model I. However, in both cases, the only species included not containing coordinated glycine are VO*+,

place in a pH region where the protonation of the ligand has no significant buffer effect, the potentiometric method can be used. To estimate formation constants, we developed programs to simulate titration curves as well as methods based on spectrophotometric results. The constants were subsequently refined using MINIQUAD3*32 and SUPERQUAD ; 33*34the results obtained with these two programs were compared. Visible isotropic spectra (VIS spectra), circular dichroism spectra (CD spectra) and EPR spectra were recorded within a wide range of amino acid and metal concentrations and amino acid: metal ratios. Spectrophotometric results are analysed by the SQUAD35-37 program. The formation constants calculated correspond to the general reaction : xVO+ +yAlaO+ zH+ Z$ (VO),(AlaO),,(H),. (1) In common with biochemical studies, we call the singly charged alaninate ion L-AlaO. We abbreviate L-alanine as HA and the alaninate ion is then A-. For the formulation (VO),(AlaO),,(H), we normally use the abbreviation M,A,,H,. For each species M&H, proposed in our final equilibrium model, possible structures are discussed. MATERIALS
Reagents

IWWW

and KW2(OH)212'.

The hydrolysis of oxovanadium(IV) has recently been reviewed.27 The nature of species present in neutral or basic solutions is still controversial but it is clear that for pH > 5 other hydrolysis products predominate which should be included in any equilibrium model, especially for systems like L-Ala +vo*+ where-even using high ligand-to-metal ratios-for pH > 7-8, solutions have a brown colour, in contrast to Set-, Thr, Cys or Asp + VO+. Two OH- : V02+ stoichiometries seem reasonable in the pH range 5-11; (i) 5: 2 (e.g. [(VO), (OH);], and (ii) 6 : 2 (e.g. [(VO),(OH)i-I,). Probably a 5 : 2 stoichiometry predominates in this pH range,27-29 although the value of n is not known (evidently it depends on the total vanadium concentration). Assuming that [(VO),(OH),]predominates in the pH range 9-12, Komura ef al. determined its formation constantin 0.1 M LiC104 and T = 25C, /320_5 = 1.4 x 10m2*. We have now studied solutions containing oxovanadium(IV) and amino acid by combining the results of potentiometric and spectroscopic techniques. Fabian and NagypS126,30have discussed the possibility and accuracy of pH-metric equilibrium studies at high ligand-to-metal ratios ; an important conclusion was that if complex formation takes * In the final calculationz9 of b2,,_ 5r K, = 1.0 x IO- I4 replaced 1.66 x lo- I4 used in the rest of that work. We recalculated fizO_ 5 taking the latter value for K,.

AND METHODS

All solutions were prepared in an inert atmosphere (high purity dinitrogen passed through sodalime and glass wool). All measurements were performed at 25.OC with solutions containing 2.25 M NaN03. Stock solutions of the VO(H,O): ion in perchlorate media were prepared by the stoichiometric addition of barium perchlorate to vanadyl sulphate (Merck) at 80C. The solutions were kept acidic by the addition of small amounts of HC104 ; normally the final HC104 concentration was around 0.1 M, this was determined by Grans procedure.3 The oxovanadium(IV) stock solutions were analysed by titration with standard KMnO,. Stock NaN03 solutions, prepared from anhydrous NaN03 (Merck pa) oven-dried at 110C for 48 h, usually contain a small amount of base and this was taken into account; similar problems were reported by Johansen and Jarns3 for KN03 (Merck pa). L-Alanine (BDH) for stock solutions was dried for 5 days (in a desiccator with silica gel in vacua). Their concentration was checked by titration with strong base. N,N-dimethyl-D-alanine (chromato-

Oxovanadium(IV)

and amino acids-I

1247

Table 1. Compositions and formation of the species in the L-Ala+VO+ system, assuming various equilibrium models, with comparable data.25s26 Values for log fl, of eq. (1) are given L-Ala+VO+ Species MLH ML MLzH2 ML2H ML, M,LzH-2 G~Y+VO~+~ Model 1I26 Model I25 10.06(0.02) 6.23(0.05) 10.96(0.06) 11.94 9.60 -6.00 -6.88 10.81(0.02) 6.51(0.03) 16.63(0.04) 11.82(0.05) 5.1(0.4) 4.1(0.06) - 1.7(?)f 1.3(0.3) -6.3(0.3) 80 - 0.7(?)f 12.11 9.64 - 6.07 -6.59 Model IX 11.16(0.01) 6.87(0.02) 21.4(0.1) 17.6(0.1) 12.4(0.1) 5.8(0.2) 4.4(0.2) -0.8(>) 12.49 9.96 -5.98 -6.95 - 22.228
h

Final (High L/M)d 11.16(0.01) 6.87(0.02) 21.4(0.2) 17.6(0.1) 12.4(0.1) 5.8(0.2) 4.4(0.2) - 1.3(0.2) - 6.7(0.2) 12.49 9.96 -5.98 -6.95 - 22.22 -

ML,H_,
MU--,

MLH-, MLH-, ML,H_ z M,LH_,

H2L

HL MH-, M&-z M&-s MH-,

a 1 M NaCIO,; 25C. 2.25 M NaNO, ; 25C ; 2 e pH e 8. From the SUPERQUAD calculation (including standard deviations) on potentiometric titrations : CH12 = 21.22. cf. CHI* = 29.93 for the model where MA2H2 is omitted, which gives values of 17.83, 12.41, 6.87, 4.77, -0.19, for the species ML2H to M,L,H_, with remaining values (MLH, H2L, MH_ ,) unchanged, as explained in the text. Other models for explaining potentiometric results are analysed in the doctoral thesis of J. Costa Pessoa (Estudos de Complexos de Oxovanadio(IV) corn Alguns Aminoacidos, Instituto Superior T&cnico, Lisbon, 1986): they all have in common the ligand species, the hydrolysed ions and the complex species MLH, ML, ML2H2, ML2H, ML*, M2L2H_, and vary only among minor species. dThese are our final values for the species formed in the systems with high L/M, self-consistent between potentiometry-model IX of this table, VIS-Table 3 and CD-Tables 3 and 4. Tomiyasu and Gordon2 included the complex resulting from dissociation of H+ from MGH (NH2 uncoordinated). Fabian and Nagypa126 included these species, of uncertain existence. g Approximated from the value of fizO_5obtained2 for a 0.1 M LiC104 medium, with correction for the ionic strength difference. When /120_5 is included in our potentiometric models as a constant to be refined, the final set of species is the same and the value refines to 10-23-10-22 with low standard deviations. hNot included in SUPERQUAD calculations : this species is certainly important for pH > 11: probably lo-* > /I,0_3 > lo-.

pure, kindly supplied by Dr S. Weinstein, Weizmann Institute of Science, Israel) and L-alanine ethyl ester hydrochloride (Aldrich) were dried for 5 days (desiccator with silica gel) before preparing their stock solutions. The NaOH solutions were prepared from Titrisol@ solutions and their concentration was determined by several graphically

titrations with standard HCl. The solutions were kept in an inert atmosphere in polyethylene bottles. Instrumentati0,r For potentiometric titrations we used a special glass vessel with a double wall, with entries for the

1248

J. COSTA

PESSOA

et al.

glass electrode (Corning 003 11 101J), calomel reference electrode (Corning 476 109 OOK), thermometer, nitrogen and base from the burette (Radiometer Autoburet ABU12). The emf measurements were made with a Corning EEL Digital 112Research pH meter. Visible spectra were recorded with a Cary 17 spectrophotometer and circular dichroism spectra with a Roussel-Jouan Dicrographe MK III using anaerobic cells. Cell compartments were kept at 25.OC with circulating water from a thermostatted bath. Unless otherwise stated, by VIS spectra we always mean a representation Of Em values vs il (E, = absorption/b x Cv, ; b = optical path) and by CD spectra a representation of A&,,, values vs 1 (As, = (differential absorption)/b x Cvo)). The X-band EPR spectra were recorded at 77 K (on glasses made by freezing solutions in liquid N,) mainly with a Bruker-ESR-ER 200tt connected to a Bruker-B-MN C5 EPR and to a Bruker EPR Data System (linked to a Nicolet 1180 computer). A few of the spectra were recorded on a Varian El09 spectrometer. MINIQUAD332 and the program used to simulate EPR spectra4 were run in an Eclipse S/140 data general computer (and a Calcomp 565 plotter) ; SQUAD35-37 and SUPERQUAD33,34 were run in a VAX 1l/780 digital computer. Calibration procedure For calibration, we assumed that the measured potential depends on [H+] according to eq. (2). The pH measuring system was calibrated by titrating dilute HCl (2-10 mmol 1-l) by standard NaOH (0.2 M), three or four times daily. For the acidic region of each titration, the Gran method was used to calculate V,. Then we calculated (i) the average ET (and standard deviation) assuming a slope S = 59.157 mV ; (ii) the Ez and S values (and standard deviations) by a linear least squares method (ET = E for the acidic region). The value of S obtained in (ii) was used to check whether the measuring system has a Nernstian slope. The MINIPOT program4 was then used to refine a value of E for the acidic region (keeping S = 59.157) ; the value obtained was considered correct for [H+] calculations. Several tests were also made for the S, JH and EO values. In the calibration procedure used for the titration curves in the LAla+VO+ system, the acid pH range was 2.3-3.3 (therefore JH = 0). The calibration was.considered satisfactory only if JH = 0 and S and Ew approximately coincide with the initial values.

E=

E+Sxlog[H+]+J,[H+]+J,,x[OH-1. (2)

For the basic region of each titration curve and using the value of E obtained in the acidic region (ET), the Gran method was used to calculate V, and (iii) K,* assuming S = 59.157 ; (iv) K,* and S by a linear least-squares method. The value of S obtained in (iv) was used to check that the measuring system has a Nernstian slope in the basic region. A constant value of -logK,* = 13.82 rtO.02 was found ; K*, is a conditional constant dependent on the apparatus and electrode used. It does not correspond exactly to the ionic product of water, K,,,, for solutions containing 2.25 M NaNO,. The MINIPOT program4 was also used to refine values for Ew, JOH, S and/or log K$. The calibration was considered satisfactory only if JOH = 0 and if S and K,* approximately coincided with the initial values. The value obtained for K,* was used in all protonation and formation constant calculations. Calibrations from data at pH 2.3-3.3 and 10.511.Omay introduce errors into results collected outside these regions. However, the use of buffers would probably introduce greater errors (since the change in liquid-junction potential due to differences in composition between buffers and test solutions may amount to several millivolts4*) and in practice, even greater errors are probably generated by the transfer of reference electrodes between solutions of different electrolytes.43 The acidity constants of L-Ala in 2.25 M NaNO, are such (pK,, 2.5 and pKti 10) that the calibration used is adequate for their determination (especially for pK,)). For the L-Ala+VO+ system, with ligand to metal ratios l-10, the calculations are based on pH values in the range 2.4-4.0 and the calibration is also adequate. However, for titration curves with high ligand-to-metal ratios (40-110) and other pH measurements in the range 3.5-9.5, our method of electrode calibration may introduce some errors. Similar approximated procedures are of course used in many other laboratories.

General experimental

conditions

Five titration curves (CAla= 3-8 x IO- 2 M) were obtained to determine the protonation constants of L-Ala in 2.25 M NaNO, ; NaOH (2 M) was used. For L-Ala+VO+, 14 titration curves were measured with initial oxovanadium(IV) concentrations in the range 1-20x low3 and L-Ala 5 x 10e4-0.45 M. Titrations involving pH measurements in the

Oxovanadium(IV) and amino acids-I range 24 and 4.5-8 were performed using ligandto-metal ratios of 0.5-12 and 48108, respectively. A total of 60 VIS spectra and 82 CD spectra (visible region) were used for the refinement of constants and calculation of spectra of individual species, with total oxovanadium(IV) and L-Ala concentrations in the range 240 x lo- 3 and 260 x lo- M, respectively. Ligand-to-metal ratios were in the range 0.5-97. RESULTS
EPR studies

1249

The EPR spectra recorded with solutions containing V02+ and L-Ala at 77 K are typical V02+ axial spectra. The region that corresponds to A,, and MI = 512 and 712gives more information about the type and number of species present. In Fig. 1, we include the high-field region of the EPR spectra of solutions with metal-to-ligand ratios of 53.9 at several values of pH. Clearly, as the pH is increased, the peaks corresponding to A,, and M, = 512 and 712 shift to lower field values (in an approximately continuous manner) until pH = 5.0. This means that in this pH range more than one species exists with rather similar A,, values (see Fig. 1). Between pH 5.0 and 6.2, one can see two distinct peaks. As the pH is increased, their relative intensities invert and for pH > 6.2 only the peaks corresponding to the species with lower A ,,are detected. The g and A (I and 11)parameters obtained by simulation of the spectra at pH 6.2 are consistent with a formulation VO(AlaO), with N202 coordination in the equatorial plane. This spectrum coincides with those of V02+ +L-Ser (or L-Thr) solutions at pH 6.0 with comparable ligand-tometal ratios. 20,21 For pH > 6.2, the spectra apparently correspond to that of only one species and (except for intensity) are very similar (at least till pH 11.3) to the one recorded at pH 6.2, only a slight shift of the MI = 512 and 712 peaks being detected. Several g and A (I and 11) values calculated by spectra simulation4 are included in Table 2. Starting with V02+ + L-Ala solutions at (a) pH 4.4 or (b) pH 4.8 with ligand-to-metal ratios of 97 and C,, = 2 x IO- 3 M, and progressively adding stock V02+ solution, the position and relative intensities of the several peaks hardly change down to ligand-to-metal ratios of 9.4 (Cvo- 1.4 x 10m2 M) and 15.8 (Cvo-9.3 x lop3 M) for (a) and (b) respectively. Similar experiments at pH 5.9 and 9.2 started with V02+ : L-Ala of 1 : 54, till respective

5.06

6.9

4000

Fig. 1. High-field range of the first derivative EPR spectra at 77 K of frozen solutions containing L-Ala and VO*+ with a L/M ratio of 53.9. The total vanadium concentration is in the range 7.1-8.7x 10m3 M. Gain = 2 x 10 and the modulation is in the range 1.6-2.5 depending on the spectra. pH values are indicated.

ratios of 15 (C,, = 0.044 M) and 11.4 (C,, = 0.05 M) were reached. The several peaks are unchanged. Except at pH 9.2, further addition of V02+ stock solution precipitated VO(OH)*. No EPR signal was detected for any solution in the region corresponding to g N 4.
Circular dichroism and visible absorption spectra

Figure 2 includes visible spectra of solutions with a ligand-to-metal ratio of 53.9. For pH < 3, except

1250

J. COSTA PESSOA et al. in a,,, (especially for 450 < 1~ 600 nm) suggests hydrolysis and/or oxidation of VO+. The CD spectra of the same solutions show enormous modifications in the pH range 1.8-12 (Figs 3, 4 and 5). Up to pH 6, the IAc,,,I values gradually increase (Fig. 6) and the I,,, of band II shifts to longer wavelengths, consistent with increased complex formation and a change in the ligand from predominantly monodentate at pH c 2.5 to predominantly bidentate for pH > 3-4. For pH > 6, the IAe,,Jvalues generally decrease except in the pH range 8-9.8 where different types of behaviour may be observed (Fig. 6). This is consistent with the gradual hydrolysis of the complexes that predominate at pH 6. For pH > 12.1, no CD spectrum is detected ; this shows that no L-Ala is then coordinated to vanadium(N). If solutions with high alanine-to-metal ratios that have been kept at pH 9-10 are acidified, equilibration is fast and the CD spectra coincide with those of freshly prepared solutions with the same pH (if dilution is not extensive), so no extensive V02+ oxidation or L-Ala racemization has occurred. However if solutions that have been kept at pH > 1l-12 are acidified, depending on the final increase

Table 2. Vanadium hype&me coupling constants and g values calculated by simulation of EPR spectra recorded at 77 K43 of solutions containing VO*++L-Ala with high ligand-to-metal ratios L/M ratio 53.9 53.9 40.0 40.0 40.0 40.0 40.0 40.0 40.0 voso,a Go (x 10) 7.88 7.50 15 15 15 15 15 15 15 -4, All (cm- x 104) 64 54 70 63 62 55 55 52 50 68 50 177 165 181 173 170 164 164 160 161 180 162

pH 3.43 8.80 1.1 2.3 4.0 6.2 6.6 10.4 13.4 1.1 13.4

g1 1.973 1.972 1.971 1.975 1.976 1.976 1.976 1.973 1.974 1.970 1.974

911 1.934 1.945 1.931 1.933 1.935 1.944 1.944 1.947 1.948 1.925 1.948

The solution contains glycerol, but no amino acid. for the a, values, the spectra are similar to the spectra of aqueous oxovanadium(IV) solutions. For pH > 3, the spectra reveal an increase of the ligand field around VO+. For pH > 6, the general

1.. :,.,;76

\
,/ .\

8.42

.\., \
.._ .. .. .\ .... . .. . . . . ..
,,

...,.\.
...\. ..\

-0

500

600 (nm)

700

800

900

Fig. 2. Visible spectra of solutions containing L-Ala+VO+ with ligand-to-metal ratios of 53.9 and Cvo = 8-10 x low3 M. The pH corresponding to each spectrum is indicated. Note the different scales on the a, axes for A, B and C. The CD spectra for the same solutions are in Figs 3 and 4.

Oxovanadium(IV) m * ,Ol x 3v *

and amino acids-I RI

0

N 1-m

\.
:

1.

\. -\ \ \

m
;: // .,
,,

\ i

\,
:

,,

. /

>

L-u

-i

3
w3v

,Ol

x %7

1252

J. COSTA PESSOA
4-

et al.

_-I / I*10.82

3/ ,/ 1 2! 5 x F 4 0, l l* I+ %, / / / 500 ,l2.1 p , _./.-.*. I -ru , -_y......_ .._... _..__...._...... 2:-l=: q-.-_._._._.-. ,/ / \ , %_ -_v_* I /-.-. / /

_._._.-.-

(Il.3 600 I

tnm)

-I -

Fig. 5. CD spectra of solutions containing L-Ala+VO+ with the ligand-to-metal ratio of 53.9 and C,, = 9-10 x 10m3 M. The pH corresponding to each spectrum is indicated. pH, the rate of equilibration varies, consistent with extensive polymerization of vanadium(IV) for pH > 12.44 For low vanadium(N) concentrations, VO(OH); would predominate.45 Figure 7 gives several examples of spectra at fixed pH (= 2.26, 2.62, 3.50 and 4.00), varying the alanine-to-vanadium ratio (0.5 to 5.0). The CD spectra change quite drastically as the total alanine concentration is increased (for low alanine-tovanadium ratios). The changes in the VIS spectra are much less pronounced. Spectra have also been recorded at fixed pH (4.4, 4.8, 5.9 and 9.2) at varying but high alanine: vanadium. The total concentration of the L-Ala in solution is constant and the concentration of V02+ is varied. At pH 4.4, the VIS and CD spectra show minor changes between alanine : vanadium ratios of 97 (Cv, = 1.8x lop3 M) and 12.1 (Cv, = 1.4x 10e2 M). For lower L/M ratios, changes in CD spectra are rather pronounced, probably due to hydrolysis of the complexes. At pH 4.8, the VIS and CD spectra gradually change (the

Fig. 6. Change with pH of the AE, values of L-Ala+V02+ solutions with a ligand-to-metal ratio of 53.9 (Figs 3-5) at three different wavelengths. (a) 697, (b) 532 [AE,x (- 1)] and (c) 637 nm.

Oxovanadium(IV) and amino acids-I

1253

Q x 3 a

0 (nm)

Fig. 7. CD spectra of solutions containing L-Ala and VO+ with Cv, = 3.340 x lo-* M. The solutions were prepared by adding the same volume of VO*+ stock solution, varying the volume of the L-Ala stock solution and adjusting the pH. The L/M ratios are indicated.

IA&,,,1 values decrease and a,,, increase) between L/M = 97 and 21, but the changes are small. For L/M < 20, the s,,,values in the VIS spectra increase drastically ; the IA&,,,1 decrease signikantly. At pH 5.9 and 9.2, the CD spectra change quite drastically as the L/M ratio varies between 55 and 20 (Fig. 8). The results in Fig. 8 can be explained only by an equilibrium, at pH 5 and 9.2, between at least two vanadium-containing species with different degrees of polymerization. Between pH 4 and 8, vanadium(IV) must be mainly coordinated to alanine, otherwise VO(OH)2 would precipitate, but the importance of a soluble oxovanadium(IV) hydrolysis product may gradually increase. Thus species with different degrees of polymerization must involve complexes of VO+ containing alanine (at, say, pH 6) but not necessarily at pH 9.2. Therefore the main features of the spectra of Fig. 8A could be explained assuming equilibria of the type :

VO(Ala0)2 (and other monomeric complexes)

L/M decreasing L/M increasing L W)dAla%(0H)2

(3)

(and other polymeric species). With L/M = 54 and Cvo = 6-10x lop3 M, while EPR spectra hardly vary in the pH range 6-l 1, the CD and VIS spectra change drastically (Figs 2-5). A possible hydrolysis product of VO(AlaO),, VO(Ala0)2(OH)-, (corresponding to VO(GlyO),(OH)considered by Fabian and Nagypalz6 as a hydrolysis product in the range 6 < pH < 8) probably has an EPR spectrum similar to that of its parent VO(AlaO)r. In fact, if the OHgroup is assumed to be equatorial, the values estimated for gr and A,,46 are 1.950 and 164.2x 10e4 cm- for VO(Ala0)2(OH)-, and 1.948 and 165.6 x low4 cm- for VO(Ala0)2 (see below). In

1254

J. COSTA PESSOA et al.

0
x

B
PH = 9.2

Fig. 8. CD spectra of solutions containing L-Ala and VO+ with Cvo = l-2.7 x lo- M (L/M ratios are indicated). The solutions were prepared by varying the volume of the V02+ stock solution; the total alanine concentration is approximately the same in all cases.

contrast, one would expect very different contributions to the CD spectra from VO(AlaO)r and VO(Ala0)2(OH)-, especially if the OH- is equatorial. In solutions containing V02+ and N,N-dimethylD-alanine with L/M = 1, as the pH is increased from 1.4 to 3.3, the IA.e,,J values gradually increase, as in similar L-Ala + VO+ solutions. For pH > 3.0, the IAE,Jvalues are substantially lower than for LAla + VO*+ solutions with the same pH, metal and ligand concentrations. With solutions containing vo*+ and L-alanine-ethyl-ester with L/M = 2,

As,,, 1: 0 in the range 400 c 3, < 800 nm between pH 1.7 and 2.3. For 2.3 e pH e 4 the JAE,,J values are approximately ten times lower than with similar L-Ala + VO*+ solutions. These experiments show that for low pH values to VO*+. (e 2.5) the NH2 group is not coordinated

Potentiometry The protonation constants calculated by the MINIQUAD and SUPERQUAD programs prac-

Oxovanadium(IV) and amino acids-I tically coincide. From MINIQUAD, for L-alanine in solutions containing 2.25 M NaNO, (at 25C) : pK,, = 2.53 and pK,, = 9.96. These values will be used in all our calculations. Formation curves calculated from our titration curves show the same trends as found by Fabian and NagypSlz6 for Gly +VO+, so we do not comment further. The system is quite complicated. Calculations involving the refinement of p, values of all possible species that may form (i.e. M,A,,H, with x, y, and z quite general) always gave results with no physical meaning. We therefore chose the following strategy for the calculations. (i) From curves of solutions with low L/M, titration points were selected to correspond to solutions where only MAH and MA (and VO*+) co-exist (several iterations were needed to select points). Taking only these these titration titration points, the MINIQUAD and SUPERQUAD programs were used to calculate the corresponding /I,, , and /I,, ,, values (the results practically coincide). These results satisfy the criteria proposed by Braibanti and colleagues.47 They also agree with those we calculated from VIS and CD spectra of solutions with L/M ratios of l-4 and 2 c pH < 3 using the MINISPEF program48 and other simple programs.** The B,, , obtained correlates well with the formation constants obtained by others49 for several monocarboxylates and for the corresponding complex with glycine.26 (ii) Taking /I,, 1and 8, ,,, as known constants, the MINIQUAD and SUPERQUAD programs were used to calculate the BxYZ of other species now including the full set of the complete titration curves. Our best model (model IX) obtained following this procedure is in Table 1. Besides the usual criteria used to select equilibrium models of speciation using these sorts of programs (CHI* and R values, standard deviations etc.), we have used a comparison with the spectroscopic results. It is this self-consistency between thermodynamic and spectroscopic results which constitutes the novelty of our method of speciation. Several other models, differing from that in Table 1 in minor respects, were acceptable if the species MA2H2 is not included. However, by including MA2H2, almost all formation constants have lower CHI and standard deviations. Moreover, calculations involving CD spectra in the pH range 2.6-3.5 also suggest that (besides MAH and MA), MA2H2 must be included to get satisfactory CD spectra for the individual species and to simulate the experimental spectra of the solutions.** For each species in Table 1, the actual values

1255

obtained in the whole set of calculations performed with MINIQUAD and SUPERQUAD lie between the maximum and minimum values presented. In model IX, we include a plausible range for each of the log/& values, established by critical examination of all calculations performed with these programs. Species important for pH > 6 show a wider range of values probably due to the uncertainty in the value assumed for /IZO-5.We emphasize that if pzO_9 is included as a constant to be refined, avalueoffi20_5 3 6x 10-23isalwaysobtainedwith a low standard deviation. The CHI* values corresponding to the models which contributed to Table 1 are relatively high (2&70). However, these values are reasonable because : the system is quite complicated ; pzo_ 5 is not accurately known ; and many different titration curves (with two different NaOH concentrations, 0.2 and 2 M) in two different pH ranges are included (2.1-4.0 and 4.5-8.0).
Spectrometry

In solutions containing VO*+ and L-amino acids, in the visible spectra, only vanadium complexes contribute to the E, or As,,, values observed. This fact removes the limitation of potentiometry to regions of pH where the ligand has no significant buffer effect. Therefore, when calculating formation constants based on CD or VIS spectra, one is in a more favourable situation for species that are important for pH > 556 because a wider range of L-Ala and VO*+ concentrations can be used as well as solutions with pH > 8. Table 3 summarizes results with the program SQUAD for solutions in the pH range 1.8-6.2. In all cases, the E (or As) values for species MAH, MA, MA2H2, MA2H and MA2 are considered as parameters to be refined. Except for /?120in CDlO, the values obtained for the constants are within the obtained in the calculations range with MINIQUAD and SUPERQUAD. Therefore one may accept as correct the values for /I, 22,/I, 2,, j?, 2. and /I**_ 2 in the last column of Table 1.
Taking B1I ,, B112, 8122, B121, 8120 and h-2 as

known constants, the VIS and CD spectra of species MAH, MA, MA2H2, MAzH and MA2 were calculated using SQUAD3S-37 based on experimental spectra in the pH range 1.8-5.1. We used a step by step procedure, following the order : VIS spectra : (i) MAH and MA,H2, (ii) MA2H, (iii) MA and (iv) MA*. CD spectra : (i) MAH, (ii) MA*H, (iii) MA, (iv) MA2H2 and (v) MA*. For example, in the calculation of the CD spectra of MA [step (iii)], after having included the CD spectra of MAH and MA2H in the input file [they were calculated in steps

1256

J. COSTA PESSOA et al.

Table 3. Calculations using the program SQUAD35,37 based on experimental VIS and CD spectra in the pH range 1.8-6.2~~ pH range of experimental spectra used in the calculations (and number of spectra included) 1.84.8 1.84.8 1.8-5.1 1.84.8 1.8-5.1 1.8-5.1 1.8-5.1 1.8-4.8 1.84.8 1.5-5.1 1.8-5.1 1.8-4.6 1.8-4.8 1.8-6.2 1.8-6.2 (23) (23) (25) (23) (25) (38) (38) (36) (36) (38) (38) (21) (36) (43) (43)

Calculation number VISl VIS2 VIS3 VIS4 VISS CD1 CD2 CD3 CD4d CD5 CD6 CD7 CD8 CD9 CD10

Number of iteration cycles 3 3 4 6 5 3 3 4 4 3 3 4 3 17 6

logB,,, (SD) 21.417 (0.084) 21.447 (0.080) 21.303 (0.095) 21.459 (0.082) 21.284 21.547 21.331 21.199 (0.042) (0.038) (0.053) (0.066)

logB,*, (SD)

logB,*, (SD)

17.632 (0.074) 17.649 (0.075)

21.261 (0.072) 21.537 (0.039) 21.280 (0.063)

17.292 17.572 17.593 17.569

(0.034) (0.018) (0.044) (0.019)

17.965 (0.014)

12.572 (0.021) 12.966 (0.018)

Unless otherwise specified the values for the formation constants were : log j?, , , = 11.16, log j?, 22 = 21.39, (VIS spectra) or AE,,,~(CD spectra) 1ogB ,,o = 6.87, logB,,, = 17.52, log/I,,, = 12.42 and lag/3,,_, = 5.80; the E.+,= values are calculated for the corresponding complex species. =The spectral region covered is 48&820 nm for VIS spectra and 447-777 nm for CD spectra (at 5.0 nm intervals). All CD data used (Aabs. and AE values) were multiplied by 10. AEON_ z not refined in the calculation. dTaking log/I,,, = 17.60 as fixed constant. These /I values are too high. Probably other species are important in the pH range 5.14.2 that have not been included, e.g. MA,H_ , and M,A,H_ 3.

(i) and (ii)], a set of experimental spectra was first chosen ; then, the CD spectra of MA, MA2H2 and MA2 were calculated using SQUAD. The values obtained for AE, ,0 were then included in the input file of SQUAD as known parameters. Step (iv) (calculation of the CD spectra of MA,H,) was then executed. The spectra so obtained were similar to those from a single calculation of all five spectra using the whole set of experimental results. The VIS spectra are in Fig. 9, the CD spectra in Fig. 10A. For pH > 6, the VIS spectra reflect the increase in importance of the hydrolysis of VO+. For pH > 8, bands I and II cannot be located. Oxidation of a small fraction of the total V(IV) may also have a substantial effect on the spectra. We therefore made no calculations with SQUAD using VIS spectra of solutions with pH > 5.1. Table 4 summarizes some results obtained with SQUAD for the CD spectra (high L/M ratios) in the high pH range. The CD spectra of M2A2H_ 2, MA,H_ ,, M2A2H_3 and MAH_, obtained in calculation CD14 are in Fig. 1OB.

DISCUSSION The proposed equilibrium model-the set of species that should be included to explain all the experimental potentiometric and spectroscopic results (with plausible values for their formation constants)---is in Table 1. For low L/M ratios, MAH- [p = 5O-150(?)] and other complex species might possibly be important. For pH > 11, VO(OH); [B = 10-18/10-19(?), probably j? N lo- *I and oligomeric species may form. Johnson and Schlemper44 identified a deepbrown species with the stoichiometry V,,O::-, which may be prepared from concentrated VOS04 solutions at pH near 14, but remarked that it is not stable in dilute solutions. The equilibrium relationship between the monomer and oligomer is not known in detail. The formation of oligomeric species at high pH (> 11-12) may account for the slow re-establishment of the equilibrium that may be observed when acidifying these solutions. The concentration distribution of the complexes

Oxovanadium(IV)

and amino acids-1

1257

(nm)

Fig. 9. Visible spectra (calculated using the SQUAD program) for complex species that may form in in solutions containing L-Ala and VO*+ (high L/M ratios) in the range 1.6 e pH e 5.1 (log /IX,,= Table 1).

formed in the L-Ala+V02+ system calculated for L/M = 53.9 and a total vanadyl concentration close to those corresponding to the EPR, VIS and CD spectra of Figs 1-5, is represented in Fig. 11. On a semi-quantitative basis, the concentration distribution shown explains the evolution of VIS and CD spectra of Figs 3-6, namely :

(i) maximum IA&,,,1 values are obtained when MA2 has a maximum concentration ; (ii) the odd behaviour of the CD spectra in the range 7.5 e pH e 9.5 may be explained by the effect of MA2 and M2A2H_2 decreasing in concentration and M2H_5 first increasing and then decreasing in concentration ;

3 2

0 x 2 z t -iI _I_

0 -I -I -2 -3

--3

\
\ L0

Fig. 10. CD spectra (calculated using the SQUAD program) for the species M,A,.H, that may form in solutions containing L-Ala and V02+ (high L/M ratios) in the pH range 1.612 (log&z in Table 1). A Species that predominate at pH < 6, B Species that predominate at pH > 6. Note the different scales for A and B.

1258

J. COSTA PESSOA et al. Table 4. Calculations of CD spectra and/or formation constants of species M,A,H_*, MA*H_ ,, M,A,H_, and MAH-, using SQUAD, based on experimental CD spectra of LAla+VO+ solutions (high L/M ratios) in the pH range 4.4-10 Calculation number

CD11 5.173 (0.011) -

CD12

CD13 5.575 (0.042) AE refined

CD14

CD15 -

log82*-* (SD) 1%PI,- 1

AE refined AE refined - 0.922 (0.019)

AErefined
4.503 (0.03 1) -1.349d (0.054) -6.71gd (0.144) 6 4.6-10.8 (20)

(SD) log/%-3 (SD)

log8,,-2 (SD)
Number of iteration cycles pH range (and number of experimental spectra included 3 4.4-6.2 (12)

AE refined 5.4096 (0.072) -

-4.569 (0.020) 4 4.610.8 (20) 7 4.6-10.8 (20)

5 4.6-10.8 (20)

The fit of the calculated and experimental spectra is not good. This probably results from not having included other species important in this pH range. bThe value obtained is too high and the CD spectrum obtained for MA*H_, is not acceptable. The p22_3 obtained seems to be high and the calculated CD spectrum is not what one would expect from the observation of the experimental spectra. dThe values obtained may be considered reasonable taking into account the experimental CD spectra and the calculations with MINIQUAD and SUPERQUAD (MAH_, is not an important species for pH < 8.0 and the /?22_-3 value is not well defined). e The value obtained is too high. If correct one would refine satisfactory p,, _ 2 values in the calculations with the MINIQUAD and SUPERQUAD. This probably results from not having included in the calculation other species important in this pH range. /Although As values (# 0) are obtained in the range 10.8 e pH e 12, experimental CD spectra in this pH range were not included owing to (i) possible formation of other hydrolysis products of V(IV), (ii) higher risk of V(IV) oxidation, (iii) increasing inaccuracy in pH measurements and (iv) sluggish equilibria.

(iii) at extreme pH values, N 1.6 and 2: 12, the As,,,values observed should be approximately proportional to BE1I, and ALE,, _ 2, respectively.

On a quantitative basis, the VIS and CD spectra calculated using SQUAD, taking the formation constants of Table 1 and the VIS and CD spectra of Figs 9 and 10, almost coincide with the corresponding experimental spectra.

Estimation offormation constants in similar systems Figure 12 shows a representation of the log j? obtained in this work for the L-Ala+VO+ system and the logfl values obtained by Fabian and

Nagypal 26 for the related system Gly +V02+ at the same temperature but in a 1 M NaC104 medium. The linear plot can be used to estimate log/3 values for MAH_ 1 and MG2H2, assuming that the values for MGH_ , and MA2H2 are correct. This type of plot (Fig. 12) can also be used to estimate formation constants of corresponding species, for example in V02+ + L-leucine, L-valine, L-phenylalanine or L-methionine systems, if the corresponding pK,, and pK,, are known in the appropriate medium. For other amino acids (like L-serine) containing other groups that may coordinate to V02+, this type of plot may also be used for species with the same stoichiometry and type of coordination. 5o

Oxovanadium(IV) and amino acids-I

1259

60 > 8 40

IO

I I

Fig. 11. Concentration distributions of the complexes formed in the VO+ +L-Ala system in solutions with C,, = 8.0 x 10e3 M and L/M = 53.9, calculated using the bxyrvalues of Table 1.
Possible structure of the complexes

Because the vanadyl ion is lop-sided, chelates of oxovanadium(IV) may form more isomers than apparently analogous mono-atomic metal centres. For example, for 1: 1 complexes of a N,Ocoordinating chelate like glycine, two enantiomers (I) and (II) may form in equal concentrations :

l-w\
H20

vHN
10

H,O,i

>

H1O

(I)

N ( II 1

In the case of L-Ala, similar 1: 1 complexes (III) and (IV), respectively, are not enantiomers ; in (III) [akin to (I)] the -CHJ is parallel to the V=O group and in (IV) it will be antiparallel. The equilibrium between (III) and (IV) is not pH dependent and unless one uses an experimental technique that does permit distinction between (III) and (IV), the determined formation constant j?l ,,, will correspond to the sum fi, 10(111) + /I 1, &V). For complexes of say L-alaninate with a MA2 stoichiometry, three isomers (V), (VI) and (VII) may form in different concentrations, again, the formation constant j?:20 corresponds to the sum B?20(V)+/%0(\1) + B:20(VW

20

=--

15 -

0 Q IO E

/
t 5,

ML

iM,L,H,

,/ ,*p+&, ,,$iALH_, I
-5 /: M2L2H-3 / , , / -5 ,

ML,%,

I
5 WI

I
IO B

I
15

I
20

tkLH_,

Fig. 12. Representation

of log /3&obtained by Fgbiln and Nagypi126 for the Gly+V02+ system in 1 M NaClO, medium and log S& obtained in this work for the L-Ala+V02+ system. The pK,, and pKaz are also represented.

1260

J. COSTA PESSOA et al.

xo/\,
N,

il

,N

CH3

HsC

O&N

N'"'0

CHs W

xo/N x
(VII 1 ~trans~

&I

CH 3

(V)

(cisl

(VI ) (rruns~

EPR, VIS and CD spectra of the isomeric species should differ. However, as the concentration ratios of (III)/(IV) and (V)/ (VI)/(VII) are not dependent on pH, CAla or Cvo, at least in a first approximation, it was not possible to distinguish the several isomers corresponding to MA and MA2 stoichiometries (nor, indeed, for other stoichiometries where different types of isomers may also be envisaged). Furthermore, in the pH range where MA2 predominates, the EPR spectra seem to involve the contribution of one species only. This could be due either to the existence of only one isomer [for example (V)] or to isomers (Vm) having very similar g and A parameters. The MAH stoichiometry probably corresponds to a complex with the R-COOgroup coordinated in an equatorial position. For the MA2H2 stoichiometry, at least two isomers may be considered, as two carboxylate groups coordinated in equatorial positions may either be cis or trans. The MA stoichiometry may correspond to structures (III) and (IV). For MA,H, as the vanadyl ion is lop-sided, four structures may be envisaged considering one L-AlaO- as bidentate and the other coordinated by the R-COOgroup (coordination involving the NH2 group is not likely). For either MA or MA2H stoichiometries, other structures may be envisaged if one considers the possibility of coordinating one of the R-COOgroups in an axial position. Using the R-COOand R-NH2 contributions tog,, and A,, ( x 1O- 4cn- ) proposed by Chasteen for additivity calculations assuming equatorial coordination, leads to 1.935,179.7 for MAH, 1.937, 176.8 for MA2H2, 1.940, 174.1 for MA and 1.942, 171.2 for MA*H. Therefore, as MAH, MA2H1, MA, MA2H (and V02+) gradually interconvert in the range 1.6 < pH < 4.0, it is not surprising that the EPR spectra in this pH region show no clear contribution from the different species. In the equilibrium model proposed, for pH > 6.2 the species that may be important are: MA,, MA2H_,, M2H2H_2, MZA2H_3, MAH_, and the hydrolysis products of V02+ ([(VO),(OH);],, VO(OH); and oligomeric species). However, as mentioned above, for pH > 6.2 the EPR spectra apparently correspond to the spectra of only one
(III) and (IV) or (VXVII)

(or two) vanadyl complex moieties and they are all very similar to the one recorded at pH 6.2 (Fig. 1 and Table 2). We will now examine briefly how this might be explained. For MA2 stoichiometry, the most plausible structures are (V)+I). Using Chasteens parameters to predict g,, and A,, for these structures, gives g,, = 1.948 and A,, = 165.6x 10e4 cm- (no correction exists differentiating cis and tram coordination). For the MA2H_, stoichiometry, if coordination involves two R-COOgroups, one R-NH2 and one OH- coordinated in the equatorial plane and Hz0 in the axial position, the prediction is similar : g,, = 1.950 and A,, = 164.2 x 10m4 cm- (if the axial position is not occupied by an Hz0 molecule, estimates of g,, and A,, are no longer safe). Therefore for such structural types, the estimated parameters approximately coincide with g,, and A,, obtained by simulating the EPR spectra in the pH range 6.2-10 (Table 2). In the case of the stoichiometries M2A2H_2 and M2A2H-3, where many different structures may be envisaged, there are two possible situations: (i) the actual structures are such that their EPR spectra are similar to those of MA2 and MA&, and (ii) these species are EPR silent. Possibly the latter explanation is correct. In fact, (VO),(OH)$ and the soluble hydrolysis species (except VO (OH);) that may be important for pH > 6 are EPR silent; 45*5 it has also been suggested that (Cu)2(AlaO)2(OH), is EPR silent.52 At pH 13.4, EPR spectra of VO*+ and LAla+VO*+ solutions with high L/M ratios practically coincide (Table 2) and no CD spectra are observed. Therefore, at this pH, L-AlaO- is not coordinated to VO*+. In the range 10 < pH < 12 the important stoichiometries are MA2H_ ,, MAH_ 2 and V(IV) hydrolysis products, and no important changes of the EPR spectra may apparently be predicted ; in fact, assuming for MAH_2 that the equatorial positions are occupied by a bidentate L-AlaO- ligand and two OH-, the estimates are g,, = 1.955 and A,, = 160.2 x 10m4 cm-, values similar to those observed for VO(OH);. As a final point, the values of molar circular dichroism for the visible transitions of the bis-

Oxovanadium(IV)

and amino acids-1

1261

G. Anderegg, E. Koch and E. Bayer, Znorg. Chim.

Acta 1987,127, 183. ;BP.A. W-%i and T. &. &a&& 1987,13& 33. Irrcng. C&m. _&a

L. F. Vilas-Boas
prebensine

and J. Costa Pessoa., in ComCbe?s_q t,~&!. Gy G.

Coordmatian

Wilkinson, R. D. Gillard and J. A. McCleverty) Sect. 33.5.9.2. Pergamon Press, Oxford (1987). #: 6: ~~I%iii, r?. 8. &n&i and I: f&h-d< hrr: J:
1974 14k, 342; Y Xsc, X Li, L. Yq c Lisr, J. Ls, on& W. c L.+ &qrlY ,Y&W&~, I,%%!,3, 55 (Chem. Abs. 1983,98, 136 555r) ; 0. F. Khodzhaev,
C&em

and L.F.V.B. thank the Instituto National de Inv&igact%o Cienti~ca e Tecnologica (INIC), Funda9ao Calouste Gulbenkian and the British Ccound for Imantia) support,
Acknowledgements---J.C.P.

U. M. Shodiev, Z. M. Musaev, Ya. S. Usmankhodzhaevaand N. A. Parpiev, tzb. Khim. Zh. 1984, 15, (Chem. Abs. 1984,102,105 09Og); U. M. Shodiev, 0. F, Xhodzhaev and N. A. Parpjev, D&I. Akad.
Nauk USSR, 1984, 35 (Chem. Abs. 1984, 103, 47

REFERENCES 1. K. Schwarz and D. B. Milne, Science 1971,174,426. 2. N. D. Chasteen, Struct. Bonding (Berlin) 1983, 53,
105.

3. K. Kustin, G. C. McLeod, T. R. Gilbert and Le Baron R. Briggs, Struct. Bonding (Berlin) 1983, 53,
139.

4. D. W. Boyd and K. Kustin, Adv. Znorg. Biochem.

1984,6, 311.

216k) ; L. Xi and Y. Xu, Kexue Tongbao (Foreign Lang. Edn), 1985, 30, 340; U. M. Shodiev, Z. M. Musaev, 0. F. Kodzhaev, Ya. S. Usmankhodzhaeva and N. A. Parpiev, Uzb. Khim. Zh. 1985,59, (Chem. Abs. 1985, 104, 156 745a). 20. R. D. Gillard, R. J. Lancashire, J. Costa Pessoa and L. Vilar-Boas, ComunicaEao 3AI1, 2 Encontro National de Quimica, Porto (1979). 21. J. Costa Pessoa and L. Vilas-Boas, 1980 Autumn Meet. Chem. Sot. University College, Cardiff (1980). 22. R. D. Gillard, J. Costa Pessoa and L. Vilas-Boas,
Comunica@o C31.9. 5 Quimica, Porto (1982). Encontro National de

5. R. L. Robson, R. R. Eady, T. H. Richardson, R. W. Miller, M. Hawkins and J. R. Postgate, Nature 1986, 322, 388 ; J. M. Arber, B. D. Dobson, R. R. Eady, P. Stevens, S. S. Hasnain, C. D. Garner and B. E. Smith, Nature 1987, 325, 372. 6. E. de Boer, Y. V. Kooyk, M. G. M. Tromp, H. Plat and R. Wever, Biochem. Biophys. Acta 1986, 869, 48 ; E. de Boer, M. G. M. Tromp, H. Plat, B. E. Krenn and R. Wever, Biochem. Biophys. Acta 1986, 872, 104; H. Vilter and D. Rehder, Znorg. Chim.
Acta 1987, 136, L7.

23. R. D. Gillard, R. J. Lancashire, J. Costa Pessoa and L. Vilas-Boas, Comunica@o PA54, 6 Encontro National de Quimica, Aveiro (1983). 24. R. D. Gillard, R. Marques, J. Costa Pessoa and L. Vilas-Boas, Comunica@o 5CP12, 9 Encontro NacionaE de Quimica, Coimbra (1986). 25. H. Tomiyasu and G. Gordon, J. Coord. Chem. 1973,
3,47.

26. I. Fabian and I. Nagypal, Znorg. Chim. Acta 1982,

62, 193.

7. R. C. Bruening, E. M. Oltz, J. Furukawa, K. Nakanishi and K. Kustin, J. Am. Chem. Sot. 1985, 107, 5298. 8. R. J. Seltzer, Chem. Engng News 1985,63,67. 9. P. Frank, R. M. K. Carlson and K. 0. Hodgson,
Znorg. Chem. 1986,25,470.

27. Ref. 18, section 33.5.5.1.2. 28. L. P. Ducret, Ann. Chim. (Paris) 1951, S12, V6, 705. 29. A. Komura, M. Hayashi and H. Imanaga, Bull.
Chem. Sot. Japan 1977,50,2927.

30. I. Fabian and I. Nagypbl, Talanta 1982,29, 71. 31. A. Sabatini, A. Vacca and P. Gans, Talanta 1974,
21, 53.

10. S. G. Brand, C. J. Hawkins and D. L. Parry, Znorg. Chem. 1987,26, 627 and refs therein. 11. R. D. Gillard and R. J. Lancashire, Phytochemistry
1984, 23, 179.

32. P. Gans, A. Sabatini and A. Vacca, Znorg. Chim.

Acta 1976, 18, 237.

33. P. Gans, A. Sabatini and A. Vacca, Znorg. Chim.

Acta 1983,79, 219. 34. P. Gans, A. Sabatini and A. Vacca, J. Chem. Sot., Dalton Trans. 1985, 1195. 35. D. J. Leggett and W. A. E. McBryde, Anal. Chem.

12. J. Felcman, M. C. T. A. Vaz and J. J. R. Frausto da Silva, Znorg. Chim. Acta 1984, 93, 101. 13. M. A. Nawi and T. L. Riechel, Znorg. Chim. Acta
1984,93, 131.

14. G. Bemski, J. Felcman, J. J. R. Frausto da Silva, I. Moura, J. J. G. Moura, M. C. T. A. Vaz and L. F. Vilas-Boas, in Frontiers in Bioinorganic Chemistry (Edited by A. V. Xavier), p.97. VCH Verlagsgesellschaft, Weinheim (1986). 15. H. Kneifel and E. Bayer, J. Am. Chem. Sot. 1986,
108, 3075.

1975,47, 1065.
36. D. J. Leggett, Anal. Chem. 1977,49, 276. 37. D. J. Leggett, S. L. Kelly, L. R. Shine, Y. T. Wu, D. Chang and K. M. Kadish, Talanta 1983,30, 579. 38. G. Gran, Acta Chem. Stand. 1950,4, 559; G. Gran, Analyst 1952, 77, 661; F. J. C. Rossotti and H. S. Rossotti, J. Chem. Ed. 1965,42, 375.

1262

J. COSTA PESSOA et al.

49. A. Lorenzotti, D. Leonesi, A. Cingolani and P. Bernardo, J. Znorg. Nucl. Chem. 1981, 43, 737 ; A.

39. E. S. Johansen and 0. Jons, Talanta 1984,31,743. 40. T. D. Smith, J. F. Boas and J. R. Pilbrow, Aust. J. Chem. 1974,27,2535. 41. F. Gaizer and A. Puskas, Talanta 1981,28, 565. 42. R. G. Bates, CRC Crit. Rev. Anal. Chem. 1981, 10, 247. 43. D. P. Brezinski, Analyst 1983,108,425. 44. G. K. Johnson and E. 0. Schlemper, J. Am. Chem. Sot. 1978,100,3645. 45. M. M. Iannuzzi and P. H. Rieger, Znorg. Chem. 1975, 14, 2895. 46. N. D. Chasteen, Biological Magnetic Resonance, Vol. 3, p. 53. Plenum Press, New York (1981). 47. A. Braibanti, F. Dallavalle, G. Mori and M. Pasquali, Gazz. Chim. Ital. 1983, 1113, 407. 48. F. Gaizer and A. Puskas, Talanta 1981, 28, 925.

Lorenzotti, D. Leonesi, A. Cingolani and A. Turolla, Gazz. Chim. Ital. 1983, 113, 229; A. Lorenzotti, D. Leonesi and A. Cingolani, Znorg. Chim. Acta 1981,
52, 149. 50. R. D. Gillard, J. Costa Pessoa and L. Vilas-Boas, to

be published. 51. J. Francavilla and N. D. Chasteen, Znorg. Chem. 1975,14,2860. 52. R. D. Gillard, R. J. Lancashire and P. OBrien,
Trans. Met. Chem. 1980,5, 340. 53. J.-P. Mathieu, Volume Commem.

Victor Henri, p. 111. Desoer, Liege (1946) ; for a later extension, see R. D. Gillard, J. Znorg. Nucl. Chem. 1964, 26, 657.