vibrational and electronic circular dichroism study of bile pigments: complexes of bilirubin and...

Analytical Biochemistry 392 (2009) 28–36

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Analytical Biochemistry

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Vibrational and electronic circular dichroism study of bile pigments:Complexes of bilirubin and biliverdin with metals

Iryna Goncharova a, Marie Urbanová b,*

a Department of Analytical Chemistry, Institute of Chemical Technology, Prague, Technická 5, 166 28 Prague 6, Czech Republicb Department of Physics and Measurements, Institute of Chemical Technology, Prague, Technická 5, 166 28 Prague 6, Czech Republic

a r t i c l e i n f o

Article history:Received 10 February 2009Available online 30 May 2009

Keywords:Bile pigmentsTransition metal chelatesCircular dichroismChiral inductionZn-required enzymes

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.05.040

* Corresponding author. Fax: +420 220444334.E-mail address: [email protected] (M. Urba

1 Abbreviations used: BR, bilirubin; BV, biliverdin;cyclodextrin; ECD, electronic circular dichroism, PLL, pcircular dichroism.

a b s t r a c t

Complexation of bilirubin (BR) and biliverdin (BV) with biogenic and toxic metals (Mn, Cu, Cd, Co, Fe, Ni,Zn, and Ag) has been studied by means of electronic circular dichroism (ECD) and vibrational circulardichroism (VCD). Poly-L-lysine and b-cyclodextrin in water were chosen as matrices capable of recogniz-ing the single stereoconformer of the pigments with defined M-helicity. Such systems allow structuralchanges caused by complexation of pigments with metals in aqueous solution at pH 10–11 to be followedusing chiroptical methods, which are intrinsically sensitive to spatial structure. These and other spectro-scopic techniques have revealed that BV and BR form monomeric complexes with Cd, Cu, and Zn anddimeric complexes with Mn. The stabilities of the complexes with Fe, Ni, Co, and Ag are remarkablylower. The sign of the ECD and VCD patterns of the complexed BV does not change for the chelates ofany of the studied metals other than Zn, this exception being interpreted in terms of manifestation ofthe opposite helicity of BV in its chelate with Zn. In the case of BR, the observed inversion of ECD signalafter complexation, together with the analysis of VCD spectra, reveals that a flattening of the moleculetakes place, i.e., an increase in the angle between the pyrrinone chromophores without an inversion ofhelicity. This chiral stereoselectivity, which is very specific in the case of the Zn chelates, is discussedin connection with the specific inhibition of Zn-required enzymes by bile pigments.

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In the body, blue-green biliverdin (BV)1 and orange bilirubin BV and BR display biological activities that frequently imply
(BR) are produced by the metabolic breakdown of heme. These lineartetrapyrroles are not planar, and in solution both exist as racemicmixtures of two isoenergetic conformers with opposite helicity. BRconsists of two conjugated dipyrrole segments joined by a labilemethylene bridge, which forms the so-called ‘‘ridge-tile” conforma-tion [1,2]. The planes of the two dipyrrinone units intersect at an an-gle of approximately 95–100�. The molecule is internally hydrogenbonded and is sparingly water soluble but highly lipid soluble andextremely cytotoxic [3,4]. BR at high concentrations may cause jaun-dice and kernicture but at the same time it shows strong antioxidantactivity [4–6].
In contrast, BV adopts a helical ‘‘lock-washer” conformation inaqueous solution, whereby contact between the terminal ketogroups is avoided [1,2,7]. This structure is stabilized by hydrogenbonding between the protonated and unprotonated nitrogens inthe ring structure. BV shows antioxidant and antiviral activity[1,6].

ll rights reserved.

nová).CD, circular dichroism; CDx,oly-L-lysine; VCD, vibrational

coordination to a metal center (M). In the complexes with metals,these linear tetrapyrroles cannot assume the M–N4 planar arrange-ment that is characteristic of metalloporphyrins [2,5,8–10]. Themetal complexes of bile pigments have helical conformations[5,7–15], which arise as a result of mutual repulsion of the twoketo groups at the terminal rings. Two degenerate conformationswith right- and left-handed helicities rapidly interconvert in solu-tion [1,14–17]. By addition of a chiral agent, the conformation ofthe complex can be driven to a preferred chirality. Conformationalspecificity induced by molecular recognition is closely related tothe functions of many biological processes, as seen, for example,in allosteric enzymes. The chirality of such systems makes it possi-ble to investigate and characterize them using techniques based oncircular dichroism (CD).

Both bile pigments are poorly soluble in water and in livingorganisms probably exist mostly bound to appropriate matrices[1,18]. They are transported to the liver as a complex with serumalbumin, bind to Cu-enzymes, and, more interestingly, as part ofbile selectively inhibit Zn-required enzymes, for example, liveralcohol dehydrogenase [18,19]. Moreover, complexes of BV andBR with proteins are capable of serving as protective agents againstmetal poisoning [18].

The possibility of chelate formation between transition metalions and bile pigments was first suggested a long time ago

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Circular dichroism of the bilirubin and biliverdin complexes / I. Goncharova, M. Urbanová / Anal. Biochem. 392 (2009) 28–36 29

[8–13,18,20–24], but the earlier data are somewhat contradictory.It has since been pointed out that the cause of these contradictionswas presumably a lack of acid–base considerations. Interactionswere mostly studied in nonaqueous media in the presence of pro-ton-binding substances, and it was concluded [8–10,15,18,21–26]that either monomeric units with helical M–N4 coordination or di-mers, in which two tetrapyrroles provide M–N4–O coordinationwith the terminal oxygen atoms acting as bridges, are formed. Itwas found [20,26] that there are three possible modes for the inter-action between metal ions and the bile pigment:

(a) metal ions form complexes by coordination of the pyrrole Natoms;

(b) metal ions are coordinated via the propionic acid side chain;(c) an ‘‘irreversible” interaction takes place presumably because

of a redox reaction.

The aim of our paper is to clarify some aspects of the conforma-tional characteristics of complexes formed by transition metalsand bile pigments in biological systems. In the current study, wehave employed electronic circular dichroism (ECD) and, for the firsttime, vibrational circular dichroism (VCD) in order to characterizethe pigments in these complexes. Moreover, we have identified theconditions that enable characterization of the metal–pigment com-plexes in aqueous media by these techniques. To elucidate the nat-ure of the interactions inducing conformational changes in a modelsystem, a flexible host with well-defined conformations was used.Two matrices were chosen for our purposes—b-cyclodextrin, as asimplified model of an enzyme binding site [27,28], and polylysinein helical conformation, as a simplified model of the serum albu-min binding [29–31]. We present herein a study on the complexesof the biogenic and toxic metals Fe, Co, Cu, Cd, Ni, Mn, Ag, and Znwith BV and BR bound to matrices in aqueous media. This studymay lead to a better understanding of the significant physiologicalroles of bile pigment metal complexes.

Materials and methods

Materials

Poly(L-lysine) hydrobromide (Mw = 44,000 g mol�1) (Sigma,USA), b-cyclodextrin (Sigma), the metal salts (Sigma/Aldrich), so-dium hydroxide (SigmaUltra, USA) and hydrochloric acid (Penta,Czech Republic) were used as received. Solutions of the matricesfor ECD study were prepared by dissolving the requisite amountin doubly-distilled water and adjusting the pH to 10.5.

Bilirubin-IXa and biliverdin-IXa hydrochloride (both from Fron-tier Scientific, USA) were used for salt preparation. Doubly-distilledwater was used as a solvent for ECD measurements. The disodiumsalts of the pigments were prepared by freeze–drying methods.Crystalline bilirubin and biliverdin were dissolved in a small excessof aqueous NaOH to ensure complete neutralization, and the solu-tions were vigorously shaken and centrifuged. All manipulationswere performed rapidly in the dark. The supernatant was frozenat the temperature of liquid nitrogen, lyophilized with protectionfrom light, and stored at �20 �C. Only freshly prepared salts wereused for the experiments. VCD spectra were recorded in D2O(99.9% D, Chemotrade, Czech Republic).

Methods

ECD spectra were recorded at room temperature on a Jasco J-810 spectropolarimeter using a quartz cell with a pathlength of0.001–1 cm; the samples were flushed with dry, ultrapurifiednitrogen before and during the experiments. A slit program that

afforded wavelength accuracy of better than 0.5 nm and an inte-gration time of 2 s for each spectral point was used. The resultsare presented as mean molecular ECD intensities with respect tothe total pigment concentration.

VCD spectra were recorded in the region 1800–1350 cm�1 atroom temperature with a resolution of 8 cm�1 using an IFS-66/SFourier-transform infrared spectrometer (Bruker, Germany)equipped with a VCD/IRRAS module PMA 37 (Bruker) by a proce-dure that has been described in Ref. [42]. A demountable cell ofpathlength 50 lm with CaF2 windows and a Teflon spacer wasused.

Sample preparation

For ECD studies, the required amounts of BR and BV salts weredissolved in matrix solutions under an inert atmosphere and inten-sively shaken. All measurements were performed within 30 minafter preparation of the complexes. Concentrations of matriceswere 10�2 M for b-cyclodextrin and 10�3 M for poly(L-lysine). Ingeneral, we worked with 1–5 � 10�5 M solutions of the pigments,which were stored in the dark. Solutions of the metals were pre-pared by dissolving the chlorides of Mn(II), Fe(II), and Co(II), thesulfates of Zn(II), Cu(II), and Ni(II), and the nitrates of Ag(I) andCd(II) in water. The chelates were obtained by addition of stocksolutions of the metal ions to solutions of the pigment–matrixcomplex to achieve the desired ratio. The final concentrations ofBV and BR were 2 � 10�5 M at pH 10.5–11.0. No precipitationwas observed in any experiment described here.

The pH values of the aqueous solutions were adjusted using di-lute sodium hydroxide or hydrochloric acid solutions and werecontrolled by means of a Cole Parmer pH meter with a glass micro-electrode (9802BN Orion). The stability of pH was verified aftereach measurement.

To examine pigment–metal interactions, a continuous variationtechnique (Job’s plot) was performed to determine the spectralchanges. For each compound, solutions containing pigment andmetal at the following molar ratios were prepared: 0:1, 1:9, 1:4,3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0. The final combined concen-tration of pigment plus metal in the mixtures was 5 � 10�5 M. Allmeasurements were performed at room temperature under anitrogen atmosphere.

CDx and metal salts used for VCD study were previously dis-solved in D2O, and the solutions were shaken for 20 min and lyoph-ilized. A solution of BV was added to a solution of CDx. Theconcentration of the matrix in samples for VCD experiments waskept at 0.1 M. The chelates were prepared by addition of the metalion solutions to solutions of the complex BV–CDx to achieve anM:BV ratio of 1:1. The final concentration of BV was 0.034 M; thepH of the solution was adjusted to 10.8 using NaOD (0.5 M solutionin D2O; Merck, USA). No correction for an isotope effect wasapplied.

All samples were allowed to equilibrate for 30 min before mak-ing the measurements, and the stability of the samples was veri-fied by comparing the infrared absorption spectra recordedbefore and after each VCD measurement.

Results and discussion

Complexes of biliverdin

ECD studyThe low energetic barrier between the P- and M-helix conform-

ers of BV leads to its rapid racemization in solution at room tem-perature [1,19,32]. Both of these conformers are able to formcomplexes with metals, and after addition of the metal ions race-

Fig. 1. ECD and UV/vis absorption spectra of biliverdin bound to b-cyclodextrin(BV–CDx) (A) and to poly(L-lysine) (BV–PLL) (B) and its chelates with Mn(III), Cu(II),Cd(II), Zn(II), and Ag(I). Inset: ECD spectra of the cyclodextrin matrix (dotted line),covalent complex CDx–Cu (broken line), and CDx–BV–Cu (full line).c(CDx) = 10�2 M, c(PLL) = 10�3 M, c(BV) = 2 � 10�5 M at pH 10.5–10.8.

30 Circular dichroism of the bilirubin and biliverdin complexes / I. Goncharova, M. Urbanová / Anal. Biochem. 392 (2009) 28–36

mic mixtures of chelates with the P- and M-conformers are alsopresent in solution. This fact prevents the characterization of bilepigments and their complexes by circular dichroism spectroscopy.However, on recognition by a chiral matrix, the racemization equi-librium is driven to a preferred conformer, and after the addition ofmetal ions a complex of preferred helicity is formed. According tothe observed ECD spectra, the metal coordination geometry in thechelates diverges from planarity but is not tetrahedral.

Chelates have previously been studied in solution, mostly inaprotic solvents, as well as in the crystal phase. Balch et al. [10]and Spasojevic and co-workers [21,22] have shown that Mn(III)forms dimers, while other metal ions, such as Zn(II), Cu(II), andCd(II), bind to BV to form monomeric complexes. The structuresof these metal–BV chelates in aprotic solvents are well established.Usually, metal ions coordinate to the doubly NH-deprotonated li-gand, with the nondeprotonated terminal ring tautomerized tothe hydroximido form (see Scheme 1). Such coordination is neces-sary in order to avoid steric hindrance between the NH group andthe metal cation. The geometry Z, Z, Z, syn, syn, syn is characteristicof all of the BV–metal complexes [7,8,11,33,34]. Highly chelatingBV and BR bind metals strongly in aqueous solution, preventingprecipitation of M(OH)n at high pH and allowing measurement ofpartial hydroxo complexes of the metals.

Fig. 1 shows ECD and absorption spectra of the complexes of BVwith b-cyclodextrin (BV–CDx) and with poly-L-lysine (BV–PLL), aswell as of their chelates with metals in aqueous solution. Additionof the metal ions leads to characteristic red shifts in the absorptionand different variations in the ECD signals that depend on the me-tal. These observations confirm that metal–BV complexes are

Scheme 1. (A) Formulas of bilirubin-IX a-ridge tile and biliverdin-IX a-helical conformations showing intramolecular hydrogen bonds. (B) Tautomerism in biliverdinstructure during chelate formation with metals.


formed at pH > 10 with the studied metals in the aqueous systemscontaining CDx and PLL, which serve as two different chiral matri-ces. Equimolar series (Job’s method) graphs (Fig. 2) show that theBV:M ratio is 1:1.

Both matrices PLL and CDx are transparent and do not show anyECD signal in region 300–900 nm. Any variation of the spectra ofalone matrixes was not induced by metal except for CDx–Cu, wherecovalent complexes are formed between Cu and AOH of cyclodextrin[35]. Spectra of CDx–Cu and CDx–BV–Cu complexes are shown inFig. 1A, inset. The characteristic signal of the CDx–Cu (broken line)was not observed in the system with BV (full line). This fact provesthe formation of the complex BV–Cu instead of CDx–Cu.

We note here that both of these matrices can only reasonably beused for conformer recognition studies in basic media (pH > 10).PLL adopts a helical conformation at this pH, which favors stereo-selective complexation with the pigments [3,4]. It was found thatstereoselective complexation of CDx with pigments also only oc-curs at higher pH. Moreover, at pH 10, BV is in the fully deproto-nated form, BV5� [11,20]. We have shown previously [28] thatBV forms complexes with CDx and polylysine mainly through elec-trostatic interaction of the ACOO� groups and that the tetrapyrroleskeleton is nonspecifically involved in the interaction. The carboxylgroups are separated from the main chromophores by two methy-lene units and therefore their ionizations are not detectable spec-trophotometrically in the UV/visible region [11,14,20]. Hence,their interaction with the OH groups of CDx does not influencethe absorption and ECD spectra of BV.

In the UV/visible region, the ECD spectra of complexes BV–CDxand BV–PLL feature two absorption bands with opposite signs: apositive band localized at 370 nm with a shoulder at 330 nm anda wide negative band at 690 nm (Fig. 1). In the complexes withboth of these matrices, the M-helical conformer of BV bindsthrough electrostatic interaction between the side propionic acidgroups, while the pyrrole moieties remain free to coordinate withthe metals [28,36]. Generally, the interaction is very sensitive toO2; the metal ions facilitate oxidation of the pigments. However,

Fig. 2. Titration of biliverdin bound to poly(L-lysine) by metal ions. (A) Equimolar sc = 5 � 10�5 M, c(PLL) = 10�3 M. (B) Spectrophotometric titration with ZnSO4. The numb

the matrices serve to retard the oxidation compared to that of un-bound BV chelates. During measurements, apparent precipitateswere not observed in the systems with matrices; however, afterabout 1–2 h, processes of oxidation were evident, and in the sys-tems with PLL, partial precipitation was observed causing strongdiminishing of absorption spectra and vanishing of CD signals.These changes were irreversible, the oxidation process were mon-itored by comparison with the spectra of the pigment metallocom-plexes without matrices in aqueous solution where oxidationprocesses started instantly.

Our working system consisting of BV bound to matrices, BV–CDx and BV–PLL, permits study of the specificity and diversity ofindividual metal complexes formed in aqueous media.

Mn was readily incorporated into the BV–CDx and BV–PLL com-plexes on treatment with MnCl2 (Fig. 1). At a metal-to-ligand ratioof 1:1, the following effects were observed, which may be ascribedto complexation. The absorption spectra of Mn–BV–CDx and Mn–BV–PLL (Fig. 1) show bathochromic shifts of both bands comparedto spectra of BV bound to CDx and PLL, respectively. The band at370 nm was shifted to �400 nm, and the band at 690 nm was sub-stantially shifted into the long-wavelength region (>900 nm). Cor-respondingly, the ECD spectra of both matrices featured a band at390 nm; the red band was substantially shifted and decreased inintensity compared to the spectra of BV–CDx and BV–PLL. Accord-ing to earlier reports [10,21,22], Mn(III) forms a complex with BVthat is likely to be of the dimeric structure, {Mn(III)BV2�}2. Thiscomplex, however, is not stable in water and could not be isolatedor extensively characterized. A monomeric form has yet to be de-tected spectrophotometrically [10,22]. Our UV/visible and ECDspectra do not provide any more detailed information. However,the VCD study presented below provides additional informationon complexes of this kind.

As previously suggested for tripyrrin-1-one chelates, the batho-chromic shifts of these chelates as compared to the free ligandcould be due to symmetry-allowed perturbations between the d-occupied metal orbital and the p-system of {Mn(III)BV2�}2

eries graphs for biliverdin bound to poly(L-lysine) and Mn, Cu, Cd, and Zn ions,ers denote the molar concentration of Zn(II), c(PLL) = 10�3 M, c(BV) = 5 � 10�5 M.


[10,22]. In the dimeric form, the flat helicoidal structure is main-tained, and one of the two terminal oxygens acts as an axial ligandof the metal of the other half of the molecule.

The complex is sensitive to oxidation; in the presence of oxy-gen, a slow metal-centered oxidation initially takes place [10,22].However, in our systems employing CDx and PLL as matrices, weobserved that the host–guest interaction between BV and thematrices prevents this olive-green complex from undergoing spon-taneous oxidation, even in aqueous solution. Under our conditions,the formed complex proved to be stable for about 1 h.

For the preparation of BV–Cu(II) complexes, decomposition ofthe pigment must be minimized. Previous studies have shown thatthe Cu complex produced is stable in aprotic solvents; however, itdecomposes in aqueous media [5,8,11,33,34]. This decompositionis accelerated by contact with air and by the presence of ionicCu2+ in the solution. This fact points to an oxidation process byO2 catalyzed by the Cu2+–Cu+ system [8,11]. The Cu complex startsto degrade as soon as it is formed. In our complex preparation,however, in which BV–CDx or BV–PLL is mixed with Cu(II) ion inaqueous solution at pH 10.5 under an inert N2 atmosphere in thedark, the green solution becomes olive within a relatively shorttime and the complexes were stabilized for the time needed to car-ry out the required experiments. The ratio dependence points tothe formation of 1:1 complexes, as was found by Job’s method(Fig. 2) described under Material and methods. The typical spectralchanges observed for the Cu complex compared to the BV matrixare depicted in Fig. 1. In the ECD spectrum, the band originally at370 nm decreased in intensity and shifted to 405 nm, the shoulderat 330 nm disappeared, and the strong negative band at 690 nmwas greatly reduced and shifted to the long-wavelength region.The chelates of Cu with both BV–CDx and BV–PLL show similarECD and UV/visible characteristics. The complex is monomeric ata 1:1 ratio and the Cu is coordinated to each of the four pyrrolenitrogen atoms in accordance with coordination chemistry de-mands. The formation of the final Cu(II)–BV chelate thus involvesa one-electron oxidation, which is allowed by the low oxidationpotential of the coordinated ligand and the presence of an unpairedelectron (d9) in Cu(II) [18,19].

In aqueous media, Cd(II) is unable to replace the NH protons ofbiliverdin [11,20], and complex formation is therefore only to beexpected at higher pH. According to our study, a 1:1 complex isformed and, taking into account its electronic structure, Cd proba-bly coordinates to BV in the same way as Cu and Zn. According toour observations and the salient literature [11,20], a monomericspecies is formed. The spectra in Fig. 1 relate to the Cd chelateswith BV–CDx and with BV–PLL. Complexation shifts both the visi-ble and the UV absorption bands to longer wavelengths. Theabsorption maximum in the UV region lies at 430 nm, and thereare two peaks at 710 and 780 nm for Cd in the visible region. Inthe ECD spectra, the changes are more pronounced: in the UV re-gion, a negative band at 327 nm and a positive band at 395 nmwith a shoulder at 360 nm are observed for the Cd complexes.Spectrophotometric titrations at various BV/Cd(II) ratios and equi-molar series graphs (Fig. 2) revealed that a 1:1 complex wasformed. The reactions proved to be reversible; that is, the spectrumof BV–CDx was restored when acid was added to the Cd complex,since complexation only occurs at pH > 10. If a solution of the Cdcomplex is left to stand in air, further spectral changes occur. Thepurpurin–Cd complex is formed as a result of oxidation of BV[18,20]. The oxidative stability of the complex Cd–BV–CDx is lowerthan is the case in nonaqueous media, but this complex is morestable than those with Mn and Cu.

The spectral characteristics of the system with Zn (Fig. 1) differgreatly from those of the complexes described so far. The spectro-photometric titration of BV–PLL with Zn2+ is shown in Fig. 2. Theobtained results indicate that BV was coordinated to Zn, but that

this process was more complex than with the other metals. Theconformation of the complex was found to be a function of pHand time, as is evident from the titration experiment. In the earlystages of the titration of BV–PLL with ZnSO4, the spectra up toc(Zn) = 1.5 � 10�5 M were very similar to those of the chelate withCd in terms of the short-wavelength pattern. The intensity of thisband was decreased and its position was shifted compared to thespectrum of BV bound to the matrix. However, the other band inthe long-wavelength region differed greatly from that of the Cdchelate: its magnitude was diminished, a weak negative patternwas observed only at 680 nm, and at the same time a positive bandappeared at 770 nm (cf. Figs. 1 and 2). Further addition of Zn led toa decrease of the positive band at 440 nm and an increase in theweak negative band at 380 nm. Simultaneously, an intensificationof the positive band in the long-wavelength region was observed.The band at 440 nm was partially inverted to a negative patternwith two maxima at 390 and 450 nm. The positive band observedat 770 nm is a result of inversion of the originally negative band at680 nm observed for BV–PLL. The Zn:BV ratio giving the maximumsignal was 1:1, and further addition of Zn did not change the ECDsignal. Our observations are in accord with previous findings [14–17] that Zn induces changes in bilidinone complexes with aminoacid esters in CH2Cl2 and that these complexes are akin to thoseformed with Cd. The complex is green and its formation is accom-panied by bathochromic shifts of the absorption bands. The ob-served bathochromic shifts of the bands point to involvement ofthe tetrapyrrole N atoms as chelating ligands. In addition, the car-boxylic groups of the pigment and/or the OH or NH2 groups of thematrices may satisfy residual coordination requirements of the Zncation. The ECD spectrum of the Zn–BV–PLL complex conjugateshowed an inversion of all of the bands compared to the spectrumof BV–PLL (Fig. 1): instead of the positive pattern, two negativebands were observed at 395 and 430 nm, and instead of the nega-tive long-wavelength pattern, a positive band was observed at762 nm.

Inversion of the ECD signal for the Zn chelate was also observedfor the complex with BV–CDx (Fig. 1A); in this system, however, incontrast to the PLL matrix, the signal changed with time. The inten-sity of the ECD bands initially decreased, and 10 min after the addi-tion of Zn(II) the ECD signal was inverted and showed no furtherchange with time. The ECD spectrum of Zn–BV–CDx shows signalsof lower intensity and only one negative maximum at 395 nm. Thecolor of the chelate changes from olive-green to brown-green. Sucha change for Zn complexes has been interpreted in terms of mono-mer–dimer equilibria [11,18,19,25]. The monomer is brown andthe dimer is green. The monomer has a distorted, square-planar,helical conformation with a hydroxo group as a fifth ligand. In the di-mer, the chromophores intertwine, sharing their Zn ions and allow-ing them to adopt an almost tetrahedral coordination geometry.Highly chelating BV binds strongly to Zn in aqueous solution, pre-venting precipitation of Zn(OH)2 at high pH and allowing character-ization of the species [Zn(BV)OH]. Zn forms five-coordinatecomplexes, with the BV ligand occupying four coordination sites[37–39] and the fifth site being occupied by water or a hydroxogroup.

An excess of Zn does not cause further variation in the absorptionor ECD signals. Zn chelates with compounds akin to BV are well de-scribed [11,18,19,25]; the fourth terminal ring of the BV ligand tau-tomerizes to form a hydroximino group (Scheme 1). The Zncomplexes are more stable than those with the other studied metals.

While chelate formation with Mn, Cd, Cu, and Zn ions leads tobathochromic shifts of the absorption and ECD bands for bothmatrices, addition of Fe and Ni causes only a reduction of the bandintensities compared to the spectra of BV–CDx and BV–PLL (spectranot shown). Ag causes a strong red shift of the absorption bandsand disappearance of the ECD signal (Fig. 1).


Addition of Ni, Fe, or Ag to BV bound to the matrices in H2Oleads to a decrease in the overall intensity without changes ofthe spectral pattern or to complete disappearance of the ECD sig-nal. For this set of chelates, the metal ions adopt coordinationgeometries that are less distorted from planarity or the complexesare less stable and probably more prone to oxidation.

Addition of CoCl2 leads to the formation of a green complex,which rapidly changes color to dark blue-green. This indicates adegradation of the BV molecule with the probable formation of apurpurin complex.

VCD studyFig. 3 shows the VCD and IR spectra of the BV–CDx complexes

with the studied metals. The high concentrations required forVCD do not permit the use of PLL as a matrix for the study of theBV chelates. This is because stereoselective complexation onlytakes place when PLL is in the helical conformation (pH > 10), butat such high pH, BV molecules bound to the PLL chain neutralizethe charge of the polypeptide and the complex precipitates.

CDx is a favorable matrix for VCD study of BV: it recognizes theM-helical conformer of BV and, furthermore, the characteristicvibrational bands of CDx are localized in the region 1450–1350 cm�1 and so do not substantially overlap with the BV bandlocalized in the region 1700–1400 cm�1, where the main variationsinduced by metal complexation are observed [28]. The IR spectrumof BV–CDx features three main bands at 1650, 1565, and1410 cm�1. The broad band at 1650 cm�1 comprises two compo-nents, of which that at �1660 cm�1 is typical for a conjugated lac-tam m(C@O) and that at 1630 cm�1 corresponds to m(C@N). As a

Fig. 3. VCD (A) and IR absorption spectra (B) of biliverdin bound to b-cyclodextrin anc(BV) = 0.034 M at pH 10.8.

result of complexation with Cu, Mn, Cd, and Zn, the m(C@O) banddisappears and the m(C@N) band is shifted to a lower wavenumberby �20 cm�1 (Fig. 3B). This indicates that the BV ligand acts as achelating agent, coordinating through the nitrogen of the AC@Ngroup. The disappearance of the m(C@O) band at 1660 cm�1 ob-served for the complexes with Cu, Cd, Mn, and Zn could indicatetautomerism of ACOANHA of the lactam form to the hydroximidoAC(OH)@N at the terminal ring of BV [7,11]. The lactam carbonylband is completely absent from the VCD spectra (Fig. 3A). For Fe,only a reduction in the magnitude of the bands at 1650 and1630 cm�1 was observed. The same was observed for Ni (spectrumnot shown). After the addition of Co, the band at 1660 cm�1 did notappear at all (spectrum not shown). Unexpectedly, in the case ofAg, this band decreased in intensity and was slightly shifted to1668 cm�1.

The bands at 1565 and �1410 cm�1 were assigned to antisym-metric and symmetric stretching vibrations of the COO� of the pro-pionic acid side groups; only slight intensity increases wereobserved in the spectra of the Cu, Zn, Mn, and Cd chelates. Smalldecreases in these absorption bands were observed for Co, Ni, Fe,and Ag. Only the antisymmetric vibration is active in the VCD spec-tra of BV bound to the matrix and its complexes with the studiedmetals. This observation proves the coordination of the metals bythe tetrapyrrole, and excludes an interaction through the carboxylgroups.

Another band located at �1500 cm�1, attributable to m(CAN) ofBV, can be expected in the spectra, and indeed a weak absorptionwas observed in the spectra of the chelates. In the spectrum ofthe metal-free system BV–CDx, this band is probably overlapped

d its chelates with Cu(II), Cd(II), Mn(III), Zn(II), Fe(III), and Ag(I). c(CDx) = 0.1 M,

Fig. 4. ECD spectra of bilirubin(BR) bound to poly(L-lysine) and its chelates withMn(III), Cd(II), Ni(II), Fe(III), Ag(I), Cu(II), and Zn(II). c(PLL) = 10�3 M,c(BR) = 2 � 10�5 M at pH 10.5. Inset: Equimolar series graph of bilirubin bound topoly(L-lysine) and Zn(II), c = 5 � 10�5 M.


with mas(COO�). In the chelates, it is shifted to lower frequencies asa result of coordination by the pyrrole N atoms. In the VCD spectraof the Cd, Cu, and Mn chelates, a corresponding negative signal isobserved at 1500–1510 cm�1, while for the Zn chelate this signalis positive.

The VCD spectra of the Cu and Cd chelates are very similar, bothbeing characterized by three negative peaks at 1560, 1550, and1510 cm�1, and differing only in slight variations in intensity.Therefore, the obtained chelates have similar structures, which isin accordance with published results on Cu and Cd complexes withBV in nonaqueous media [11,19,20]. Cu and Cd form complexes inthe +2 oxidation state. Cu has very limited coordination chemistrybecause the MIId9 complex has little tendency to add axial ligandsand is only able to bind a single donor ligand to form a pentacoor-dinate complex. From the existence of ECD and VCD signals, it isevident that BV is not planar in the metal complexes; otherwise,no signals would be observed. The changes in these spectra arein accordance with the fact that distortion of the central BV chro-mophore is less than that in the ‘‘lock-washer” conformation, butthe previous helicity of the pigment is nevertheless retained onchelate formation. BV probably forms mononuclear, four-coordi-nate MN4 complexes with Cu(II), whereby the ligand forms a heli-cal unit in which the two end groups on the tetrapyrrole overlap.BV cannot be planar; the metal ion adopts a coordination geometrythat is distorted from planarity, but is certainly not tetrahedral.Thus, the complex probably has the form of a distorted square pyr-amid. As for Cd, the dominant process in solution is the formationof a five-coordinated adduct, that is, a square-pyramidal Cd ioncoordinated by nonplanar helical BV.

The VCD spectrum of the Mn complex displays two characteris-tic negative strong bands, a broad, strong band at 1560 cm�1 and aless intense band at 1510 cm�1. Compared to the VCD signals of theCu and Cd chelates, the band at 1560 cm�1 is stronger. It is as-signed to an antisymmetric COO� stretching vibration. The ob-served intensity increase indicates a better defined position ofthese groups in the chelate, the structure of which has to undergogreater adjustment. Moreover, from the IR spectra we concludethat the COO� groups are not involved in the chelate formation,so their adjusted position may be purely a result of the adjustedposition of the two tetrapyrrole moieties in the chelate. Our con-clusions are in agreement with the fact that the Mn(III) complexhas the dimeric structure {Mn(III)BV2�}2, as was found from X-ray diffraction studies [10].

Although the IR spectral pattern of the Zn–BV chelate is similarto the spectra of the above-noted chelates, the VCD pattern showsdistinct differences. The band at 1560 cm�1 shows positive sign,i.e., opposite compared to the signals of the other chelates, andthe other bands are diminished. This observation is in accordancewith the ECD data (cf. Fig. 1A and B), which were indicative of aninversion of chirality for the brown-green Zn chelate.

For the Ni, Ag, and Fe chelates, no shift of the C@O stretchingband compared to the spectrum of the metal-free BV–CDx was ob-served, indicating that tautomerization does not take place in thesechelates. In the VCD spectra, only a decrease of the main bandscompared to the spectrum of BV–CDx was observed (shown onlyfor the Fe chelate).

The Co complex was found to be highly susceptible to oxidationat higher concentrations; in air, it is very readily converted to otherproducts and a VCD signal is no longer observed.

Complexes of bilirubin

ECD studyPrevious studies on the formation of metal–BR complexes have

mostly been conducted in nonaqueous solvents in the presence ofproton-binding substances, whereby coordination was found to in-

volve the pyrrole N atoms [5,10,18,20,23,24,26,40,41]. ECD spectraand equimolar series graphs of the chelates of BR bound to PLL inaqueous solution are shown in Fig. 4. A two-step mechanism hasbeen proposed for the complex formation [41]: a metal ion be-comes coordinated to one dipyrrole chromophore of the BR mole-cule and this coordination is followed by chelation of the secondmetal ion by the remaining half of the molecule. Electrochemicalstudies [24] have indicated that BR can also form complexes withCd, Fe, and Zn in water at pH 8, but their structural characterizationhas been hampered by their instability. It has been reported[18,20,23,26] that the interaction between BR and metals in aque-ous solution results in pigment degradation, in which BR is con-verted into BV.

Of the two matrices that we used, complexes on the polypep-tide matrix PLL are more stable compared with those on CDx, onwhich the pigment degrades very rapidly. Its oxidative stability isdistinctly lower compared to BV–PLL. We have shown that thePLL matrix plays a key role in photostabilization, not only for theBR molecule but also for BR chelates. It has enabled us to studythe conformational changes of BR bound to PLL in chelates withmetals in aqueous solution for the first time.

The absorption and ECD spectra of BR–PLL and its chelates withmetals in alkaline solution at pH 10.5 are shown in Fig. 4. Job’splots showed the metal-to-BR ratio of the chelates with BR–PLLto be 2:1 (shown for the Zn complex only). The characteristic redshift observed in the absorption spectra indicates that the processof complexation involves the dipyrrinone ‘‘halves” acting as chelat-ing ligands. In addition, the carboxylic acid groups may satisfy theresidual coordination requirements of the metal cations.

Additions of Cd, Mn, Ni, and Fe (Fig. 4) led only to decreasedintensities and minor changes of the main bands of the BR–PLLcomplex at 530 and 440 nm in the ECD spectra, and decreasedintensities and a slight shift of the band to longer wavelengths inthe absorption spectra. However, the pH of the solutions may not


have been high enough for the metal ion to induce deprotonationof the pyrrole NH groups (with pK > 14). Therefore, in the studiedpH range of 10.5–11.0, the complexes may only have been partiallyformed. At higher pH, BR complexes are rapidly and irreversiblyoxidized to other pigments.

On the other hand, addition of Ag or Cu to the system led to asubstantial decrease in the band intensity of the BR couplet inthe ECD spectra and to a blue shift of the absorption bands.

On addition of Zn, the absorption band of BR was shifted to alonger wavelength (not shown) and the orange-yellow solution be-came red. A Job’s plot showed that the Zn-to-BR ratio of the chelatewith BR–PLL was 2:1. The most remarkable feature was that theECD signal was inverted; a negative couplet was observed afterthe addition of Zn to the BR–PLL system. However, the complexwas less stable compared to its analogue with BV.

Our overall set of spectral data on the complexes of BV and BRwith Zn complexes reveals that both of these pigments form com-plexes with Zn and that the ECD spectra were opposite to those ofthese pigments bound on matrices and also opposite to those of theother metal chelates. Therefore, the structure of the pigment–Znchelate must differ substantially from those of the other metalcomplexes. This was found for both of the studied matrices, PLLand CDx. There is more than one possible interpretation of theseresults. For BV, the signal inversion may be attributed to the oppo-site helicity of BV in the chelates with Zn [1,2,7,36]. Our study hasshown that complex formation of BV with Zn takes place in twosteps—initial formation of the olive-green complex with spectralproperties similar to those of the Cd chelates, followed by inversionof helicity and formation of the brown-green chelate. Taking intoaccount the fact that even in nonaqueous solution compounds akinto BV prefer P-helicity in their complexes with Zn [14–16], we canconclude that inversion of the ECD and VCD signals is a result ofopposite helicity of the pigment. In the case of BR, rationalizationof the obtained spectra is more difficult. Inversion of the ECD spec-tra of BR may occur in two ways, by inversion of the helicity of thepigment or by the flattening of the molecule, i.e., an increase in theangle between the pyrrinone chromophores without inversion ofhelicity [1,4,27,36]. Support for one of the interpretations was pro-vided by a VCD study of the M–BR chelates.

VCD studyFig. 5 shows the VCD and IR spectra of BR bound to PLL and its

chelates with Zn and Cd. The observed positive couplet at1650 cm�1, which is typical of the a-helical conformation, indi-

Fig. 5. VCD (A) and IR (B) spectra of bilirubin (BR) bound to poly(L-lysine) and itschelates with Zn(II) and Cd(II).

cates that PLL adopts the helical conformation in the complexeswith BR, even at high pH and at the high concentrations neededfor VCD. However, an overlapping of the absorption bands of thepigment with the amide I and II absorption bands of PLL hampersspectral study of the chelates in such systems. The CDx matrix thatwe also used to study chelates in aqueous solution is not useful forthis system because the process of BR oxidation after metal addi-tion is very fast.

The IR spectrum of BR–PLL features three broad bands at 1650,1560, and 1410 cm�1. A broad band at 1650 cm�1 comprises char-acteristic amide I vibrations of the polypeptide, the pigmentm(C@O), the vinyl-coupled lactam, the pyrrole ring stretching,and the pyrrole ring deformation, and is characteristic of moleculespossessing intramolecular hydrogen bonds.

As a result of complexation with Cd or Zn, this broad absorptionband was diminished; the amide I vibration modes were not chan-ged, but the pigment modes changed significantly. The lactam andpyrrole vibrations were shifted toward lower wavenumbers by�20 cm�1 and were seen as a shoulder at 1580 cm�1. This observa-tion may be rationalized in terms of two dipyrrinone units beinginvolved in the chelation; the BR ligand acts as a chelating agentcoordinating through the pyrrole nitrogens. Moreover, in view ofthe high sensitivity of VCD to the local structure of chiral molecules[28,42], the similarity of the changes observed for both Cd and Znchelates indicates that the complexation mode in these chelatesmust be very similar.

As regards the broad band at 1600–1530 cm�1, it consistsmainly of the asymmetric COO� stretching vibrations of the propi-onic groups in the BR structure (1560 cm�1) and the pyrrole C@C,NAH, and lactam CAN stretching vibrations at 1545 cm�1. Wehave previously observed the corresponding VCD signal in the sys-tem BR–CDx. The antisymmetric stretching vibration of COO� at1565 cm�1 is active in the VCD spectra of BR and its chelates,and can be used as a characteristic signal for the determinationof the helicity of BR [28].

The VCD spectra of BR–PLL, Cd–BR–PLL, and Zn–BR–PLL are verysimilar, with only a small shift of the positive peak at 1560 cm�1

being observed for the chelates compared to BR–PLL. Therefore,the obtained chelates have the same helicity of the BR part in allof these complexes. The VCD results thus support the interpreta-tion that BR adopts a more open conformation in the chelate withZn and that inversion of the pigment helicity does not take place.Taking together all of the obtained facts, we can interpret ourobservations on Zn–BR in terms of a characteristic flattening ofthe molecule that causes inversion of the ECD signals of BR che-lates with Zn.

Conclusion

The results of this study have shown a great difference in thestructural characteristics of Zn chelates and those of other biogenicmetals. BV and BR form monomeric complexes with Cd, Cu, and Znions and dimeric complexes with Mn. Complexes with Fe, Ni, Co,and Ag have not been described because of their low stability.The helicity of the pigments did not change for any of the studiedmetals other than Zn, with which inversion of the main ECD andVCD patterns was observed. For BV, the inversion of the ECD andVCD signals can be attributed to opposite helicity of this pigmentin the chelates with Zn. Our study has shown that complexationof BV with Zn takes place in two steps—initial formation of an ol-ive-green complex with spectral properties similar to those ofthe Cd chelate, followed by rapid inversion of the helicity and for-mation of the brown-green chelate. In the case of BR, the obtainedspectra may be rationalized in terms of a flattening of the mole-cule, i.e., an increase in the angle between the pyrrinone chro-mophores without an inversion of helicity.


These features may result in the bile pigments displaying a spe-cific ability to inhibit Zn-required enzymes. Both of these pigmentsare good chelating agents and probably inactivate Zn-metalloen-zymes by binding to their Zn atoms. Inhibition occurs by way ofthe formation of a mixed enzyme–metal–pigment complex orthrough the formation of an enzyme–metal–substrate–pigmentcomplex. In this case, the chelating agent has to satisfy the criteriaof stereoselectivity to achieve a conformation that is appropriatefor complexation at the active centers of the enzyme. The unusualcharacteristics of the Zn chelates of BR and BV may play a crucialrole in understanding these processes.

Acknowledgments

This work was supported by research grants from the Ministryof Education, Youth, and Sports of the Czech Republic (MSM6046137307) and from the Grant Agency of the Academy of Sci-ences of the Czech Republic (IAA 400550702).

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