07) 210–216www.elsevier.com/locate/geoderma

Geoderma 142 (20

Spectroscopic approach for elucidation of structural peculiarities of Andisolsoil humic acid fractionated by SEC-PAGE setup

Claire Richard a,⁎, Ghislain Guyot a, Agnès Rivaton a, Olga Trubetskaya b, Oleg Trubetskoj c,Luciano Cavani d, Claudio Ciavatta d

a Laboratoire de Photochimie Moléculaire et Macromoléculaire, UMR CNRS-Université Blaise Pascal 6505, 63177 Aubière Cedex, Franceb Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia

c Institute of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russiad Dip. di Scienze e Tecnologie Agroambientali, Alma Mater Studiorum - Università di Bologna, viale G. Fanin, 40, I-40127 Bologna, Italy

Received 27 April 2007; received in revised form 20 July 2007; accepted 19 August 2007Available online 21 September 2007

Abstract

A Andisol humic acid (HA) was fractioned by preparative size exclusion chromatography and polyacrylamide gel electrophoresis in thepresence of denaturising agent (SEC-PAGE setup). Three fractions exhibiting high, medium and low molecular size (MS) and showing well-defined and distinct electrophoretic mobility were obtained. These fractions were analysed by UV–visible, FTIR and 3-D fluorescencespectroscopies. UV–visible absorbance and A465/A665 ratio of fractions increased as the MS decreased. FTIR measurements coupled with HCltreatment of samples revealed that aliphatic moieties such as polysaccharides and peptides were mainly localized in high MS fractions whilecarboxylic functions in low MS fractions. Excitation-emission matrix spectra showed that long-wavelength emissions were almost exclusivelyobserved upon excitation of low MS fraction. These data indicate that fractionation by SEC-PAGE setup yielded pools of soil HA macromoleculesshowing distinct structural characteristics.© 2007 Elsevier B.V. All rights reserved.

Keywords: Humic substances; Fractionation; SEC-PAGE; FTIR; Excitation-emission matrix

1. Introduction

Structure and chemical composition of humic substances(HS), operationally divided into humic acids (HAs, insoluble inacid) and fulvic acids (FAs, soluble in both acidic and alkalinemedia), may vary depending on their pedo-climatic origin andage. Their detailed molecular structure still remain unclear andno data have shown convincingly whether HS are cross-linkedmacromolecules or loosely held aggregates. Another veryimportant question concerns chemical components distributionbetween HS of different molecular sizes (MS). Owing to theimportant role played by HS in mobility and fate of plantnutrients, regulation and environmental contaminants, it isparticularly useful to better characterize them. On the other handtheir heterogeneity in MS and charges make this task extremely

⁎ Corresponding author. Tel.: +33 473407142; fax: +33 473407700.E-mail address: claire.richard@univ-bpclermont.fr (C. Richard).

0016-7061/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.geoderma.2007.08.019

difficult (Abbt-Braun et al., 2004). Among chromatographicmethods that are ordinary used for HS fractionation, techniquesbased on the size-exclusion effect appear to be most useful, asthey allow to relate elution data on HS MS distribution (DeNobili and Chen, 1999; Muller et al., 2000; Janoš, 2003).However, one of the main problems that often arises during sizeexclusion chromatography (SEC) fractionation is interactionbetween HS and gel matrix, mainly through hydrogen bonds(Janson, 1967; De Nobili and Chen, 1999). Electophoreticmethods provide detailed HS characterization as well (Schmitt-Kopplin et al., 1998; Janoš, 2003), but it is very difficult torelate the electrophoretic data to other fractionation methods ofHS.

In prior studies (Trubetskoj et al., 1997) we have developedan effective method for the fractionation of soil humic acids(HAs) based on the combined use of SEC on Sephadex G-75and polyacrylamide gel electrophoresis (PAGE). This procedureis called SEC-PAGE. The novelty and advantage of this

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combination rests primarily on the presence of urea, whichadded at a level of 7 M both in SEC and PAGE assists in therapture of hydrogen bonds and prevents interaction between thefractionated humic material and the solid immobile phase onwhich substances are separated (Wallqvist et al., 1998). This isaccompanied by the disaggregation of HA, a process, webelieve, is primarily related to the disruption of hydrogen bonds.The proposed procedure allows separation of primary disag-gregated structural humic components, thereby solving some ofthe key problems occurring in fractionation of HS, e.g.,irreversible/reversible chromatographic column adsorption andinfluence of PAGE chemical components (e.g. tris and/or borateions) on the HA fractions composition. Using PAGE incombination with SEC makes possible to obtain preparativeamounts of fractions differing in both electrophoretic mobility(EM) and MS.

This technique was first applied to HAs extracted fromseveral soils of European part of Russian Federation. Prelim-inary analyses showed that high MS fractions contained higherconcentration of amino acids (Trubetskaya et al., 1998) andwere much less absorbing than low MS fractions (Trubetskojet al., 1999). Later on, fractions of different soil HAs and of astandard HA of the International Humic Substances Society(IHSS) were tested for their fluorescence and photoinductiveproperties (Trubetskaya et al., 2002; Richard et al., 2004). It wasfound that the low MS fractions contained most of fluorophoresand most of compounds capable of degrading phenolic probesunder light excitation (Richard et al., 2004). The data veryrecently obtained by thermochemolysis with subsequentidentification of products by gas chromatography/mass spec-trometry showed that regardless of the HA origin SEC-PAGEfractions have been enriched in different structural componentswhose distribution was independent of HA genesis (Saiz-Jimenez et al., 2006).

In the present work, we applied the SEC-PAGE set-up on aHA extracted from an Andisol soil (France). We obtained threefractions showing distinct MS and EM which were analysed byUV–visible, FTIR and fluorescence spectroscopies for a bettercharacterization.

2. Experimental procedures

2.1. Materials

The soil sample was taken from the A horizon of naturalAndisol (Aurillac, France, 44.56°N). The main physical–chemical properties were the following: total organic car-bon=2.9%, CEC (cmolc kg−1)=22.3, pHH2O=6.2. The soilsample was air-dried and ground to pass through a 2 mm sieve.Before extraction, plant debris were removed by flotation. TheHAwas then extracted using IHSS extraction procedure (www.ihss.gatech.edu). Water was purified using a Milli-Q (Millipore)device. Disodium hydrogenophosphate and potassium dihy-drogenophosphate were 99% purity and supplied by Prolabo(France). The C, H, N-analysis of HA was performed on aPerkin Elmer CHN Analyzer, series II 2400. Elemental analysesvalues were corrected for water and ash contents that were

measured on a Perkin Elmer Thermal Analyzer at 105° for theformer and 550 °C for the latter. Analyses were preformed intwo replicates. We found 56.1% for C, 5.8% for H and 7.3% forN. The oxygen content can be estimated to be around 30% asthe difference from 100%. The content of ash was 3.8%. Thesedata are typical for terrestrial HAs (Visser, 1983).

2.2. Fractionation

Fractionation of soil Andisol HA by setup SEC-PAGE (i.e.preparative fractionation by SEC monitored by PAGE) waspreviously reported (Trubetskoj et al., 1997). Briefly: 10 mg HAwas dissolved in 1 ml 7 M urea and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5x100 cm), equilibratedwith the same solution. Void (Vo) and total (Vt) column volumeswere 47 and 160 mL, respectively. Void volume was determinedusing Dextran Blue 2000. Flow rate was 15 mL h−1. The UV-detector (ISCO, USA) was set at 280 nm. Column effluent wascollected as 2 mL aliquots using collector fraction (ISCO, USA)and each aliquot was assayed by PAGE according to Trubetskojet al. (1997). The apparatus was a vertical electrophoresisdevice (LKB 2001 Vertical Electrophoresis, Sweden) with gelslab (20×20 cm). Electrophoresis was carried out in 10% PAGat the room temperature for 1 h at a current intensity of 25 mA.As the gel buffer 89 mM Tris-borate, pH 8.3, with 1 mM EDTAand 7 M urea was used. The sample buffer (0.05 mL) contained89 mM Tris-borate, pH 8.3, 7 M urea, 1% sodium dodecylsulfate and 1 mM EDTA. On the basis of PAGE analysis, threefractions were obtained from HA sample. Fractions weredialyzed seven days against distilled water (cut-off of thedialysis membrane was 5000 Da), lyophilized, and used forfurther physical–chemical analyses. For obtaining preparativequantities of fractions, fractionation procedure was repeated 20times.

The weight of each fraction was calculated by directweighting after chromatography. The weight distribution offractions was measured using the ratio Wi/ΣWi, where Wi is theweight of fraction and ΣWi the weight of all fractions obtainedafter SEC fractionation of HA.

The comparison of the absorbances at 280 nm before andafter chromatography was made in a separate experiment andshowed that sorption of HA on Sephadex G-75 gel matrix wasnegligible, more than 98% of applied HA were collected afterelution. Briefly: one portion (10 mg) HA was dissolved in200 ml 7 M urea and optical density of the solution at 280 nm(A280 before chromatography) was detected. Another 10 mg of HAsample was fractionated on Sephadex G-75 as described in theprevious paragraph, all aliquots from Vo to Vt were combinedinto the whole volume, dilute to 200 ml with 7 M urea andoptical density at 280 nm (A280 after chromatography) was measured.The ratio A280 after chromatography/A280 before chromatography wasmore than 0.98.

2.3. Amino-acid analysis

Approximately 2–4 mg of HA and fractions were hydrolysedwith 5.7 M HCl for 24 h at 110° C under nitrogen atmosphere.

Fig. 2. UV–visible spectra of bulk Andisol HA and its H–MS, M–MS and L–MS fractions. Normalized spectra at 50 mg L−1 in phosphate buffer (pH 6.5).

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Released amino acids were determined using analyzer LC 5001(Biotronic, Germany). Analyses were repeated three times sothat the standard deviations were less than 5%. Analyzercalibration was carried out using amino acid standards (PiersStandard H, 16 amino acids at 2.5 μmol ml−1 +Cys at1.25 μmol ml−1). The percentages of total amino acids (w/w)were 13.0±0.5, 16.5±0.8, 13.1±0.3 and 6.0±0.2 for bulk HA,and high, medium and low MS fractions, respectively.

2.4. Spectroscopic analysis

UV–vis spectra were recorded on a Cary 3 (Varian)spectrophotometer in a 1-cm quartz cuvette. HAwas solubilizedunder stirring in Milli-Q water containing phosphate buffer pH6.5 (6.6×10–3 mol L−1). All the solutions were filtrated on0.45 μm Millipore filters prior to use. Excitation EmissionMatrix (EEM) spectra were recorded on a Perkin–Elmer LS-55luminescence spectrometer that was equipped with a xenonexcitation source. Excitation and emission slits were set to 10and 5 nm band pass, respectively. To limit second order ofRayleigh scattering, Raman diffusion and fluorescence emis-sion, a 290 nm cutoff filter was used. Correction forinstrumental configuration was made using the calibrationdata for excitation and emission factors provided by themanufacturer. When necessary, and in order to minimize theexcitation inner filter effect, solutions were diluted to obtain anabsorbance equal to 0.100±0.005 at 300 nm for all the samples.The emission wavelength range was 300–700 nm and that ofexcitation 250–500 nm. Stepwise increments of 1 and 10 nmwere used for emission and excitation wavelengths, respective-ly. Infrared spectra were recorded using a Nicolet 5SXC-FTIRspectrometer equipped with a thunderdome-ATR (4 cm−1 and128 scans). The thunderdome is a single reflexion accessoryequipped with a germanium crystal. Acidic treatment ofsamples was made by placing samples in an atmospheresaturated with HCl vapor. This treatment selectively converts

Fig. 1. A) Electrophoresis of 0.15 mg Andisol HA in 10% polyacrylamide gelin the presence of denaturing agents. B) Size exclusion chromatography of5 mg Andisol HA on Sephadex G-75 column (100×1.5 cm) using 7 M urea aseluent. Black boxes on the X-axis show the fractions obtained on the basisof polyacrylamide gel electrophoresis: 1 : H–MS fraction, 2 : M–MS fraction,3 : L–MS fraction.

carboxylate species (CO2- ) into corresponding carboxylic acid

(CO2H) that shows a different IR absorption (Avram andMateescu, 1970; Lin-Vien et al., 1991).

3. Results and discussion

3.1. HA fractionation

Electrophoresis and SEC profile of bulk HA is shown inFig. 1. Three main bands appear in the electropherogram. Thefraction that does not move into the 10% polyacrylamide gelcorresponds to humic matter eluted in the excluded volume ofthe column. This fraction which shows the highest MS wasnamed H–MS. The fraction in the mid part of the gelcorresponds to HA eluted in the elution volume 58–80 mland was named medium MS fraction (M–MS). At last, thefraction on the bottom of the polyacrylamide gel corresponds tothe HA eluted at the end of total column volume (110–158 ml)was named low MS fraction (L–MS). The distribution is 27±3%, 18±2% and 25±2% for high, medium and low MSfractions, respectively. The difference to 100% that correspondsto the remaining fraction is mainly constituted by a mixture offractions M–MS and L–MS.

3.2. UV–visible spectroscopy

The UV–visible spectra of HA and fractions are shown inFig. 2. Absorption spectrum of bulk HA is typical for soil HAs,showing a monotonous decrease with increasing wavelengthand a shoulder around 280–300 nm. Absorbance varies in the

Table 1Absorption coefficients of bulk HA and its fractions at 280 nm (A280) and A465/A665 and A250/A365 ratios

Sample A280 (L g−1 cm−1) A465/A665 A250/A365

Bulk HA 20.4 6.7 2.95High MS fraction 9.1 4.3 2.87Medium MS fraction 13.9 4.8 2.79Low MS fraction 26.6 9.5 3.02

Fig. 4. FTIR spectra of bulk Andisol HA and its fractions after HCl treatment.

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order: H–MSbM–MSbbulk HAbL–MS showing that ab-sorbing moieties are not randomly distributed among fractionsbut concentrated in smaller size fractions. Table 1 listsabsorption coefficients at 280 nm (A280) that were computedby dividing absorbances at 280 nm (A280) by the concentrationof organic matter expressed in g l-1. Value for low MS fractionsis almost three-fold higher than that for high MS fractions.Absorbance in this wavelength range is commonly attributed toπ−π⁎ electron transitions in aromatic or poly-aromaticcompounds (Chin et al., 1994; Peuravuori and Pihlaja, 1997).The pattern as that described in Table 1 suggests that thefractions with the lowest molecular sizes hold a much greaterquantity of complex unsaturated bond systems than the fractionswith the highest molecular sizes. The A465/A665 and A250/A365

ratios for each fraction are also given in Table 1. The first ratio isoften used in soil science as an indicator of humification: aprogressive humification leads to a decrease of the A465/A665

ratio. In our case, the ratio is higher for low MS fractions thanfor high or mediumMS fractions as generally reported whateverthe used fractionation procedure (Christl et al., 2000; Duarteet al., 2003). The second one is traditionally related to thepercentage of aromaticity and the molecular size of humicsubstances. For instance, this ratio was satisfactorily applied ona series of aquatic humic solutes to estimate size andaromaticity; when the ratio A250/A365 increased the aromaticity

Fig. 3. FTIR spectra of bulk Andisol HA and its H–MS, M–MS and L–MSfractions. Part A: full spectra and zoom in the 3000 cm−1 spectral range in insert.Part B: zoom in the 1900–900 cm−1 spectral range.

and molecular size of aquatic humic solutes decreased(Peuravuori and Pihlaja, 1997). However, in our case, theratio does not significantly vary from a fraction to the other,probably because aromaticity and molecular size of fractionsshow opposite variations.

3.3. ATR–FTIR spectroscopy

The FTIR spectra of Andisol HA and fractions are shown inFig. 3. Part A gives the full spectra and the zoom in the 3000–2800 cm−1 spectral range and part B the 1900–900 cm−1

spectral range where the observed differences are the largest.Bulk Andisol HA exhibits the typical absorption bands of HAs.We observed a broad band in the 3600–2700 cm−1 spectralrange due to OH and NH vibrations and a small band around2950 cm−1 corresponding to aliphatic stretching vibrations. Inthe region extending from 1800 to 1500 cm−1, several maximaappear. A maximum is observed at 1651 cm−1 and shouldersaround 1700, 1595 and 1530 cm−1. Frequencies in this IRregion are related to ν(C=C) stretching vibrations of aromaticrings and to ν(C=O) stretching vibration of carbonyl containingfunctions. The band of CO2

- appears around 1590 cm−1 whilethat of CO2H in the range 1700–1710 cm−1. Between 1600 and1680 cm−1 are generally observed ν(C=O) stretching vibra-tions of quinones and those of conjugated or H-bondedcarbonyls. For instance, amides show ν(C=O) stretchingvibrations in this frequency range (amide I band). This bandis generally associated with a band at lower frequencies (1500–1560 cm−1 for secondary amides) corresponding to the N–Hbending vibration (amide II band). It must be pointed out that ashoulder appeared in Andisol HA spectra around 1550 cm−1

which may correspond to the amide II band. The spectrum ofFig. 3 exhibits a second massif between 1470 and 1320 cm−1.This region is related to aliphatic groups (C–H symmetricbending of methyl groups of aliphatic chains), to ν(C–O)stretching vibration of CO2

− (1386 cm−1) and of phenols as wellas to O–H bending of alcohols and carboxylic groups. Twoother bands peaking at 1250 and 1040 cm−1 are observed. Inthe 970–1250 cm−1 appear ν(C–O) stretching vibrations of

Fig. 5. Excitation Emission Matrix spectra of bulk Andisol HA and its H–MS,M–MS and L–MS fractions.

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alcohols, ethers and CO2H as well as vibration of minerals, forinstance SiO2 at 1130 cm−1.

To make easier bands assignment, we treated Andisol HAwith HCl in order to acidify carboxylate functions. As expected,this treatment vanishes the 1590 and 1386 cm−1 bands anddrastically increases the 1700 and 1255 cm−1 bands thatbecame prominent (Fig. 4). The band at 1651 cm−1 remainsunchanged after acidic treatment but appears as a shoulder ofintense 1700 cm−1 band. After HCl treatment, the band at1530 cm−1 is much more evident. The presence of both 1651and 1530 cm−1 bands might correspond to secondary amides.But, the 1651 cm−1 band can also be assigned to quinones andaromatic rings.

Spectra of fractions reveals significant structural differences:

1/ The band around 2950 cm−1 corresponding to aliphaticstretching vibrations is more intense in H–MS than in bulkHA. It is almost absent in L–MS and M–MS fractions. Thus,H–MS fraction appears to be much more aliphatic than theother fractions.

2/ Without HCl treatment, L–MS fraction exhibits a main peakat 1595 cm−1 and a shoulder at 1650 cm−1. In contrast, H–MS and M–MS fractions are dominated by an intenseabsorption with maximum at 1651 cm−1. The HCl treatmentaffects fraction spectra diversely. H–MS fraction is poorlyaffected, in contrast to M–MS and L–MS fractions for whichthe 1595 cm−1 band is replaced by the 1700 cm−1 band.Moreover, the band at 1386 cm−1 also disappears in L–MSand M–MS. It can be thus concluded that H–MS fractionscontained a much smaller concentration of carboxylatefunctions than L–MS and M–MS. The greatest change dueto the HCl treatment is observed for L–MS fraction thatshould be the fraction containing the highest concentration ofcarboxylate functions.

3/ The shoulder around 1530 cm−1 is clearly visible in H–MSandM–MS but absent in L–MS. After the HCl treatment, theregion around 1500–1550 cm−1 is free of carboxylateabsorption making easier the observation of this band at1530 cm−1. It is only present in H–MS and M–MSsuggesting that polypeptidic fonctions are present in muchhigher concentration in H–MS and M–MS than in L–MS.

4/ The intensity of the 1040 cm−1 band drastically decreaseswith MS. This band corresponds to C–O vibrations of ethersor alcohols and can be attributed to carbohydrates. Thus onecan conclude that their concentration decreases with MS.

3.4. Fluorescence spectroscopy

The excitation-emission matrix (EEM) spectra of AndisolHA and its fractions were recorded on solutions showing all anabsorbance of 0.1 at 300 nm in order to minimize inner filtereffect and to get an emission intensity proportional to theemitting center number. Spectra are presented in Fig. 5. BulkHA exhibits a broad emission band with a maximum at 500–510 nm and a short-wavelength emission with a maximum at340 nm. Aweak emission around 525 nm is also observed. Thisfeature is typical for soil HAs (Senesi et al., 1991; Alberts and

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Takacš, 2004). The emission (λexc/λem: 280 nm /350 nm) isgenerally attributed to proteinic constituents (tryptophan-like)(Coble, 1996). The broad emission band (λexc/λem: 250 nm–400 nm/380 nm–550 nm) results from conjugated systems, theemission wavelength depending highly on the degree ofconjugation, on substitution and on the presence of heteroatoms(Coble, 1996). The EEM spectra of fractions drastically differsfrom that of bulk HA. The λexc/λem: 280 nm/350 nm emission ismore intense in H–MS than in the other fractions, while thelong-wavelength emission is mainly observed in L–MS. Lastly,the weak emission with maximum at 525 nm can be observeddistinctly in the L–MS fraction.

4. Discussion

The obtained spectroscopic data on bulk HA and its SEC-PAGE fractions give valuable information on the distribution ofsome specific constituents between them. From FTIR analysesof HA and its fractions before and after HCl treatment, we coulddistinctly visualize aliphatic C–H, carboxylic acids and ethers.Some good indication concerning amides were also obtained.Low MS fraction is enriched in carboxylic groups compared tothe other fractions and especially to high MS fraction. Such anincrease of carboxylic acid content with a decrease of MS wasalready reported in previous studies using various fractionationprocedures, SEC (Swift et al., 1992; Li et al., 2003) or ultrafiltration (Wang et al., 1990; Christl et al., 2000; Francioso et al.,2002), but not in a so clear-cut way. High MS fraction wasfound to concentrate a great part of aliphatic C–H, amides andethers, these functionalities being poorly present in low MSfraction. The increase of amide content with the increasing ofMS as detected by FTIR fits perfectly well with the increase ofamino-acids concentration among fractions obtained by aminoacids analysis. These data are also in line with the increase ofaliphatic character with the increasing of MS mentioned byChristl et al. (2000) and Li et al. (2003). Concerning the fact thathigh MS fraction is concentrated in peptides and carbohydrates,it is interesting to note that Watt et al. (1996) observed asignificant correlation between amino acid and carbohydratecontents in HS, the amino acid content being about 1.7 timeshigher than the carbohydrate content.

UV–visible data reveals that low MS fraction shows higherabsorption than high MS fraction. It seems to be directlyconnected with a lower concentration of non-absorbingfunctionalities such as polysaccharides and amides in theformer and to a higher concentration of aromatic compounds asindicated by the absorbance data at 280 nm.

Fluorescence properties were also found to vary drasticallywith the MS of fractions. The fluorescence emission 280 nm/340 nm (λexc/λem) that can be assigned to protein-like materialsand tryptophan is more intense in high MS fraction than in theother fractions. This result is in line with the distribution foundfor amino acids among fractions and the observation of amide inH–MS but not in L–MS. On the other hand, the long-wavelength emitting fluorophores are almost exclusivelylocated in low MS fractions. It has been proposed that thisemission is related to conjugated systems bearing electro-

withdrawing groups such as carboxyl, carbonyl or Schiff-basestructures (Senesi, 1990; Senesi et al., 1991; Alberts andTakacš, 2004). Our FTIR data that shows the enrichment oflow MS fraction in carboxylate functions validate theseassumptions.

The results presented in this paper and very recently obtaineddata (Saiz-Jimenez et al., 2006; Sanchez-Cortes et al., 2006)bring evidence that the distribution of components among SEC-PAGE fractions is not random. It could suggest the existence ofbasically different structures in soil HAs which could beseparated by the SEC-PAGE fractionation in the presence ofdenaturing agents. It is in accordance with the recentunderstanding of conformational structure of humic substancesthat suggests supramolecular associations of relatively smallmolecules stabilized by hydrophobic associations, chargeinteractions, hydrogen bonds and metal bridging (Piccolo,2001; Simpson et al., 2002).

Acknowledgements

The work has been supported by INTAS grant 06-8055 andRussian Foundation for Basic Research (project 06-04-48266-a).

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