Revista Mexicano. de FÚlica 38, Suplemento 1 (!992) 160-16li

Eleetroehemieal studies on sorne Cu-Mo interaetions.n. Synthesis and electroehemieal behavior oí the

Histidyl-histidine-Cu eomplex*CECILIA ,IUÁREZ-GoRDIANO

Instituto de Investigaciones BiomédicasUniversidad Nacional Autónoma de México

Apartado postal 70-228, 04510 México, D. F., México

TOMÁS HERNÁNDEZ P., IGNACIO GONZÁLEZ

Depar.tamento de QuímicaUniversidad Autónoma Metropolitana-Iztapalapa

Apartado postal {j5~534, 093.{0 México, D.F., MéxicoAND

ANTONIO Qumoz-GuTIÉIlREZ

[n.,tituto de Física, Universidad Nacional Autónoma de MéxicoApartado Postal 20-364, 01000 México, D.F., México

Hecibido el 5 de sepliembre de 1991; aceplado el 31 de enero de 1992

A BSTRACT. HistidyI rcsiducs are important ligands for the copper ion in rnan}' IIlctailoprotcinswith biologicaJ activities, but rarely two His-rcsidues occur consecutively in the same protein. \Vesynthesized the Hi3tidyl-histidine dipeptide (JI-JI) in solnlion (classical way), using dicyclohexyl-carbodiimide as coupling reagent. The JI-!l-Cu cornplex was prepared wilh lhe JI-l! dipeplide indyrnethylsulfoxide (DMSO) solution in a molar ratio of 3:1 with CuSO,. Triangular and inversevoltammetry over a platinum eleetrode were carried out both on the dipeptide and the complex. Bythe obtained voltammograms it was observed that the JI-JI dipeplide induced Cn deposition on lheelectrode. Since the !I-JI dipeptide was electrochemically inactive, their capacily to accnmnlate Cnon the electrode can be utilized for quantitative copper assays, even in difrerent copper oxidationstates.

RESUMEN. Los residuos histidil son importantes ligandos del ion cobre en muchas rnctaloproteÍnasde importancia capital en biología, sin embargo, raramente se encuentran dos residuos de histidinaconsecutivos en la misma proteína. Sintetizamos el dipéptido Histidil-histidina, en solución porel método clásico utilizando diciclohexilcarbodiimida como agente acoplante, posteriormenteprepararnos el complejo !I-!l-Cu utilizando el dipéptido en solución en dimetilsulfóxido en unradio molar de 3:1 con CUS04. Una vez obtenido el complejo, estudiamos su comportamientoelectroquímico mediante voltametrÍas inversa y triangular, llevadas a cabo sobre un electrodode platino. En los voltamogramas obtenidos pndimos observar qne el dipéplido lIistidil-hislidinafavorece el depósito de Cu sobre la superficie del electrodo, además de que es inactivo y no produceseñales electroquímicas propias; capacidades por las que proponemos su utilización en métodos deanálisis cuantitativo de Cu y aun en diferentes estados de oxidación de este metal de transición.

PACS: 82.80.Fk; 87.22.Fy

'S"pporled by SETECOMFIA-uAM-Azcapotzalco.

ELECTROCIIEMICAL STUDlES ON SOME Cu-Mo INTERACTIONS. 11... 161

Blue copperNormal Copper ---

3000

1000

24.000 16.000 8.000 2500 2900 3300

Energy (cm1) -Field (gauss)

FIGURE 1. Comparison oC the optical and EPR spectra oC a bine Cn site and normal tetragonalCu ceuter. lu the EPR spectra the blue copper site exhibits a smaller copper hyperfine splittingand one intense absorption band at about 600 nm.

l. INTRODUCTlON

The eopper proteins are associated with a variety of biological funetions sueh as oxygentransport, eleetron transfer and free radicals control as in the superoxide dismutase [1].Copper proteins are unique coordination compounds since the protein ligands imposeunusual eoordinatiou properties, which are not present in inorganic complexes and modelcompouuds. So geometrie and c!ectronic structme of the Cu atoms at the active site are aconsequence of orbital deformations caused by protein dynamics which very often inducedistortions as the Jahn- Teller effect (Fig. 1).The principal coordination modes of copper ions to proteins is through the thiol groups

of cysteine and the imidazole moiety of histidine, since the coordination atoms preferred bycopper are the sulfur, nitrogen and oxygen [2,3). The imidazole groups of histidine residueslocalized at the active si tes of many enzymes act aS general acid-base catalysts in a numberof key biochemical readions. Ilistidine containing small peptides are present in plants andanimals in which are connected with metal metabolism, acting as growth factors or theirreceptors [4,5). The tripeptide Glycil-histidyl-Iysin (GIlL) complexed with Cu found inhuman serum, is a growth factor used for in vitro culture of a diverse variety of cells andorgans ['1]. In addilion lhe GIlL-Cu complex has significanl superoxide dismutase-likeaelivily and has beeu assoeialed wilh tissue-proteetive, anti-trauma, augiogenesis anduemogenesis effects [51.The Il-Il-Cu eomplex has been used in medicine in the treatment of deficient states

as the ~tcBkc's dis('a.~("'.sinc(' this complcx is v('ry similar lo the physiological transportform of Cu(!!) in blood serum [6,71. And the active si te of hemocyanin is a lIistidine-Cucomplex in whieh some histidines are dominant and others are distant, giving to the CuamI O atoms the parlieular spatial arrangements required for the hemocyauin funetion [81.

162 CECILIA JUÁREZ-GORDIANO ET AL.

The synthesis of a short oligoglycine peptide catalyzed by H-H in prebiotic conditionswas achieved since the eighties [9]. The prebiotic synthesis first of histidine, and laterof the H-H dipeptide under cyclic wet-dry laboratory conditions, consistent with theevaporating prebiotic ponds, was reported [10,11]. The enhancement activities of H-Ilin promoting prebiotic reactions involving nucleotide derivatives and oligonucleotides asdephosphorylation of dAMP, hydrolysis of oligo(A)12, and the oligomerization of 2',3'-cAMP was recently demonstrated. AH this supports the hypothesis that simple prebioticpeptides containing at least two imidazole groups was involved in the prebiotic formationof other peptides and nucleic acid molecules and these were the preceding events in theprebioUc origin of enzyme biosynthesis [121. The interactions between histidine and metalions has been extensively studied in solution but an abnormal behavior of Cu and histidinehas being reported in aH cases [13,14]. Histidine complexes with metals are more staolethan the corresponding metal complexes of glycine, alanine and valine [15]. This may bedue to the existence of chelating ~oups in histidine, other than amino and carooxilategroups, probably the nitrogen atoms in the histidine imidazole are lhe preferent ligandsfor the Cu ion in the proteins, this is also indicated by absorption spectra in the ultravioletrange [16].However severa! studies show that the carboxylate group of histidine should be involved

in the metal bonding, since after cheJation the amino group is no longer titratable, proo-ably indicating that the amino group is involved in chelation [14,17]. CrystaHographicstudies of the Cu-His complex (grow up at pE 3.7) have shown two histidine moleculescoordinate with a central Cu atom, each one through the amino nitrogen and "carboxylateoxygen, but the imidazole groups are not coordinated to the eu atom and tum away fromit. This show that histidine is able to coordinate through its amino and carboxylate groups,thus behaving as a "normal amino acid" [14].Poly-L-histidine binds Cu at pll values higher than 3.0 with a maximum binding con-

stant of 1 X 1019 mol-1 at pH 5.0 in this complex, the Cu(Il) ion is oound to threeimidazole rings and a peptide nitrogen [18,19]. More recently, poly-L-histidine was shownto be strongly adsorbed at mercury electrodes and to be a suitable reagent for modifyingelectrodes and concentrating Cu [13].Although histidyl residues are important metal ion binding sites in proteins, only rarely

two His-residues occur conseculively in lhe same protein chain [20J. This may be so for asteric hindrance, or by rigidity in these molecules, and that is way the chemical synthesisof dipeptides H-H is not easy and the yield is poor [21]. It is important to emphasize thatMerrified's first papers mention that the imidazole ring was an inhibitor of the peptidegrowing chain, during the peptide synthesis [22]. \Ve synthesized the E-Il dipeptide andits Cu complex to study and characterize its electrochemical behavior.

2. EXPERIt-.1ENTAL

The 1Ii3tidyI-histidine dipeptide synthesis was carried out by solution in the classicalway [23]. AH chemicals and solvents were of analytical degree. Trifluor-acetic acid (AldrichChemical Company Inc.), dichloromethane and ethyl acetate (Sigma Chemical CompanyInc.) were distilled with sodium carbonate prior to use. Amino acid L-Ilistidine and dicy-

ELECTROCIIEMICAL STUDlES ON SOME Cu-Mo INTERACTlONS. n... 163

59.8wÜz~f-~ "4(f)Z<{a:f-.,J¿ 3;19o

<&1).0 limO m>.0 2200.0 IlllO I400D lXll.O ~.oo ~.oo 400.00

WAVENUMBERS [cm -1]

FIGURE 2. IR spectrum from the product obtained by the chemical synthesis. The presenee ofamide group (-NlI-Ca-) with -NII- at 1680 cm-1 and C=O at 3310 cm-I indicates the peptidicbond formation, therdore the II-II dipeptide was obtained.

c1ohexylcarbodiimide (Sigma Chemical Comp. Ine.) and BoeIlis Tosyl was from PeninsulaLab. Ine. A solution of BoeHis Tos, (409.2 mg, 1 mmol) in diehlorometane (20 mi) waseooled in an ice water bath and treated with dieyclohexylcarbodiimide (412 mg, 2 mmol)after that with a solution of L-lIistidine (304.4 mg, 2 mmol) and triethylamine (5 mi).Stirring and eooling (O°C) was eontinued for about one hour and the reaetion mixture wasat room temperature for 24 hours. After the reaetion mixture was filtered and the residuewas in ethyl aeetate (50 mi), the solution was extracted with a mixture of HCI O.IN-NaCI (1:1 v/v). Then it was washed with aqueous solution of NallCOJ-I1aCI (1:1 v/v)and saturated solution of NaCI, then was coneentrated in vacuo to dryness. A dry whitepowder was obtained [24].The deproteetion step was earried out by dissolving the solid of Boe-Bis-Bis in tri-

fiuoraeetie acid (50 mi) at room temperature for one hour. After that the solution wasevaporated to dryness in vacuo, the solid fraetion was washed with diehloromethane (twotimes) and dried in vacuo. The produet was extracted in the usual manner [231, and wasmilled to powder with dry ether and. dried to solid in vacuo. The prod uet was tested f."amide group formation by IR speetroscopy (Figure 2). And the thin layer ehromatographyover Siliea Gel showed one spot.The I1-I1-Cu eomplex was made in situ using the ll-ll dipeptide with a solution of

CUS04 in DMSO in a molar ratio of 3:1. The mixture was neutralized with 0.1 N NaOH.Deeantation was enough for removing the insoluble material, usually exeess nf eopper(11) as its hydroxides [4], the eomplex formation was demonstrated by UV /Vis speetrum(Figure 3).The linear sweeps of inverse and triangular voltammetry was earried out on a plat-

inum eleetrode as working eleetrode, a glassy earhon har as auxiliary eleetrode, and a

164 CECILIAJUÁREZ-G,oRDIANOET AL.

2.0

(f)m«

1.0

200W

400 650 900WAVELENGTH

FIGURE 3. Optica) absol'ption spectrum of Il-II-Cu complex in DMSO. The band's shift on thespeetra (from 322 to 382 nm) for the Cu-DMSO when 11-11dipeptide was added, indicates thecomplex formation.

-0.62V,

+0.62V

A

-O.66V +O.66V

B

FIGURE4. Typieal triangular s,,"eepvoltammograms on piatinum e1ectrode of CUSO, (5 X 10-5 M)in 0.1 M/DMSO. SW<'CPrate 50 mVs-1 A) In ahsence of Il-Il dipeptide, ll) in presenee of 11.11dipeptide.

Ag/ AgN03 modified electrode, as the reference e1ectrode in tetraethylammoniulIl bromide0.1 1vI.A Princeton Applied nesearch Model-273 wa." used. This studies on t.he H-H-CIlcomplex were made in DiviSO in a Ilon-sleady stat.e regime as has bee" <kscribed [251.

ELECTROCIIEMICALSTUDIESONSOMECu-Mo INTERACTIONS.n... 165

-O.76V +O.76V

A

-O.74V +O.74V

B

FIGURE5. Linear sweep inverse voltammograms on platinum eleclrode oC CuSO, (5 x 10-' M) in0.1 M TEAB/DMSO, sweep rale 50 mVs-l. The tirsl anodie sweep (~) was perCormed arter coppere1eclrodeposilion al -1.0 V. A) In absence oC H-H dipeplide, B) In presence oC H-H dipeplide.

3. RESULTS AND DISCUSSION

In figure 4a lhe behavior of CuSO, in a 5 X 10-' M solulion is shown. The anodic peak at-0.33 V is due lo copper oxidation, as may be observed lhis peak is very smal!. 1I0wever,in lhe figure 4b we can observe lhe resull of an experimenl done in lhe same condilions inwhich the H-H dipeptide has been added in a 5 x 10-5 M solution, and lhis voltammogramshows a bigger anodic peak at -0.31 V. This peak may represent Cu oxidation, but as maybe.observed the peak current increases. It is probable that the H-H dipeptide accumulateCu on the platinum electrode.To prove that the anodic peak at -0.30 V was related to Cu oxidation, we increased the

H-H dipeptide in the same experimental conditions, then the Cu deposit at the electrodeincreased as can be seen in the vollammogram of figure 5b in which the same -0.37 Vpeak is larger than in figure 5a (from 14.67 /lA to 25.54¡lA). This was another indieationthat the dipeptide concentrate Cu at the electrode.Since the same experiment shows that the II-H dipeptide is electrochemieally inert

(the isolated H-H dipeptide does nol produce any signal), this dipeptide can be used forconcentrating and detecting small amounts oC Cu ions in well defined oxidation states andtheir possible changes with chelating agents, such as the tetrathiomolybdate.

REFERENCES

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2. R. Lontie, C/in. Chim. Aeta 3 (19.58)68.3. J.C. Moreira, R. Zhao and A.C. Fogg, Ana/y.! 115 (1990) 1561.

166 CECILIA JUÁREZ-GORDIANO ET AL.

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3054.17. M.A. Doran, S. Chaverek and A.E. Marlell, J. Amer. Chem. Soe. 86 (1963) 2129.18. M. Palumbo, A. Cosani, M. Terbojevich and E. Peggion, Maeromoleeules 11 (1978) 1271.19. A. Levilzki, T.I. Peeh and A. Berger, J. Am. Chem. Soe. 94 (1972) 6844.20. R.P. Agarwal and D.D. Perrin, J. Chem. Soe. Dalton 89-92 (1976).21. M.H.V. Van Regenmorlel, J.P. Briand, S. Muller and S. Plave, Synthetie peptides as antigens.

Laboralory Techniques in Bioehemislry and Molecular Biology. Elsevier, Amslerdam (1990).22. D. Krieger, B.W. Erickson and R.B. Merrifield, Proe. Natl. Aead. Sei. USA 73 (1976) 3160.23. M. Bodansky and A. Bodansky, The praetiee o/ peptide synthesis. Springer-Verlag, New York

(1984).24. J.M. Slewarl and J.D. Young, So/id phase peptide synthesis. Freeman Sei. G. (1969).25. A.J. Bard and L.R. Faulkner, Eleetrochemieal Methoda. Fundamentals and Applieations. John

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