Activation of hydrogen peroxide by copper(II) complexes with some histidine-containing peptides and their SOD-like activities

Activation of Hydrogen Peroxide by Copper Complexes with Some Histidine-Containing Peptides and Their SOD-Like Activities Jun-ichi Ueda, Toshihiko Ozawa, Makiko Miyazaki, and Yumiko Fujiwara J-IU, TO. NationalInstituteof RadiologicalSciences, Chiba-shi,Japan.-MM, College of Pharmacy, Tokyo, Japan

YF. Kyoritsu

ABSTRACT The reactivities of copper complexes with histidine-containing oligopeptides towards active oxygen species such as hydrogen peroxide (H,O,) and superoxide ion (0;) were investigated by electron spin resonance (ESR)_spin trapping and thiobarbituric acid WA) methods. At physiological pH values, Cu(I1) complexes with oligopeptides containing histidyl residue at the N-terminal could more easily activate H,O, to yield hydroxyl radical (-OH) than other Cut10oligopeptide complexes containing histidyl residue in the second or third position. Further, it was suggested that since all Cu(II)-oligopeptide complexes examined could scavenge O;, these complexes have SOD-like activities.

INTRODUCTION Active oxygen species such as superoxide (0;) being formed by leakage of electrons to oxygen (0,) from various components of the cellular electron transport chains and provided during the respiratory burst of phagocytic cells, have been implicated both in the aging process and in degenerative diseases, including arthritis and cancer [1,2]. Therefore, the biological system possesses the protective mechanisms against active oxygen species. For example, 0; is dismutated to O2 and hydrogen peroxide (H,O,) by superoxide dismutase (SOD), and H,O, thus formed is converted to 0, and water by catalase or

Address reprint requests and correspondence to: Dr. Toshihiko Ozawa, National Institute of Radiological Sciences, 9-l Auagawa 4-chome, Inage-lot, Chiba-shi 263, Japan. Journal of InorganicBiochemist,

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glutathione peroxidase. Therefore, SOD has a beneficial effect as an antiinflammatory agent and is used in the therapy of many diseases [2-61. However, it has recently been suggested that Cu,Zn-SOD activates H,O, to give a more reactive radical species, hydroxyl radical (*OH) [7-141. In order to confirm the conversion of H,O, to *OH catalyzed by Cu,Zn-SOD, we have investigated the reactions of H,O, with SOD 1151and its model compounds in detail by use of ESR-spin trapping and thiobarbituric acid (TBA) methods. In this paper we report SOD model compounds such as Cu(I1) complexes with peptides containing histidyl residue at the N-terminal strongly scavenge 0; and also activate H,O, to give -OH. EXPERIMENTAL Materials L-histidylglycine (HisGly) and glycyl-L-histidine (GlyHis) were purchased from Sigma Chemical Co. Ghistidyl-Ghistidine (HisHis) was purchased from Kokusan Chemical Co. Glycylglycyl-L-histidine (GIyGlyHis) and glycylglycyl-Lhistidylglycine (GlyGlyHisGly) were purchased from Bachem Chemical Co. (HisGlyGly), glycyl-L-histidylglycine Other peptides, Lhistidyl-glycylglycine (GlyHisGly), Ghistidyl-Lhistidylglycylglycine (HisHisGlyGly), and L-histidylglycyl-Ghistidylglycine (HisGlyHisGly) were synthesized by classical peptide chemistry in solution [16]. TBA, hypoxanthine (I-IPX), SOD, and xanthine oxidase (XOD) were purchased from Sigma Chemical Co. 5,5Dimethyl1-pyrroline N-oxide (DMPO) was purchased from LABOTECK and used without further purification. Chelex 100 resin (sodium form) was obtained from Bio-Rad Co. Preparation

of Reaction Solutions of C&I)

Complexes, H,O,, and 0;

Cu(II1 complexes were prepared by the addition of 1.1 times higher concentrations of ligands to copper(B) ions. The concentration of H,O, in aqueous solutions was determined by titration with 0.10 M KMnO,. 0; was generated by the HPX (2mM)/XOD (0.4 U/ml) system at pH 7.4 in 0.1 M phosphate buffer. Deionized and triply distilled water was treated with Chelex 100 resin. ESR Measurements ESR measurements were carried out on a JEOL JES-RE-1X ESR spectrometer (X-band) with 100 kHz field modulation. ESR spectra were recorded at room temperature in a JEOL flat quartz cell. ESR parameters were calibrated by comparison with a standard Mn*‘/MgO marker. Further, the magnetic field was calibrated with an NMR fieldmeter (JEOL ES-F(S). Detection of TBA-Reactive Substance (TBARS) TBARS with oxidation products of deoxyribose by OH radical was determined according to the described method [17,181. The standard reaction procedure was as follows: Cu(I1) complex (0.2 cm3, 1 mmol) was added to deoxyribose (0.5 cm3, 7.5 mmol), together with distilled water (0.9 cm31. Further, 0.2 cm3 of substrate or distilled water was added followed by 0.2 cm3 H,O, solutions (100 mmol). Samples were incubated at 37°C for 1 hr and thiobarbiturate reactivity devel-

ACTIVATION

OF HYDROGEN

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BY Cu(II) COMPLEXES

125

oped following the addition of thiobarbituric acid (1 cm3) cl%, w/v in 0.05 M NaOH) and glacial acetic acid (1 cm3). The sample tubes were heated for 15 mm at lOO”C, cooled, and the absorbance at 532 nm was read. Spectral Measurements Visible absorption spectra were recorded on a Union Giken SM-401 spectrophotometer and a Hitachi U-3210 spectrophotometer at room temperature. RESULTS AND DISCUSSION 1. Interactions of C&I)-Ollgopeptlde

Complexes with Hydrogen Peroxide

Table 1 shows the results obtained by the TBA method for the reactions of some C&I) complexes with H,O,. From Table 1 it is apparent that C&I) complexes of oligopeptides containing histidyl residue at the N-terminal greatly accelerated the generation of TBARS, whereas Cu(I1) complexes of oligopeptides containing histidyl residue in the second or third position slightly increased. These facts indicate that C&I) complexes of oligopeptides containing histidyl residue at the N-terminal have high reactivities towards H,Oz to yield more reactive -OH, although those containing histidyl residue in the second or third position slightly react with H,O,. In order to ascertain the formation of -OH from the systems mentioned above, reactions of Cu(II)-oligopeptide complexes with H20, were investigated by ESR spectroscopy using DMPO as a spin trap. The ESR spectrum similar to DMPO-OH adduct (aN(l) = aH(l) = 1.49 mT) was observed during the reactions of C&I) complexes of histidine-containing peptides with H,OZ (Fig. la). The formation of -OH was confirmed by the occurrence of the 1-hydroxyethyl radical (CH,CH,OH) adduct of DMPO (aN(l) = 1.58 mT, aH(l) = 2.28 mT) by addition of ethyl alcohol to Cu(II)-H,O, reaction systems as shown in Figure lb. These results were summarized in Table 2. The strong signal intensity of DMPO-OH was obtained from the reaction of H,02 with Cu(I1) complexes of oligopeptides containing histidyl residue at the N-terminal (except Cu(II)HisGlyHisGly), whereas the weak or no signal intensity of DMPO-OH was observed from the reactions of H,O, with C&I) complexes of oligopeptides containing histidyl residue in the second or third position. From the results TABLE 1. Formation of TBA-Reactive Species (TBARS) from the Reaction of Cu(II)Histidine Oligopeptide Complexes with H,O, Copper011 Complexes

TBARS blmol/ml)

Cu(II)_HisGly Cu(II>-GIyHis cll(II)_HisHis CuUI~HisGlyGly Cu(II~GlyHisGly CdII~GlyGIyHis Cu(II~HisGlyGlyGly Cu(II)_GlyGlyHisGly Cu(II)_HisHisGIyGly Cu(IIMIisGlyHisGIy

152.5 19.7 167.6 145.0 6.4 10.8 166.1 19.7 190.9 10.4

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I

1mT

FIGURE 1. ESR spectrum observed from the reaction of Cu(II)-HisGly with hydrogen peroxide in the presence of DMPO. (a) Cu(II)+IisGly (0.25 mM) + H,O, (25 mM) + DMPO (25 mM); (b) Cu(II)-HisGly (0.25 mM) + H,O, (25 mM) + 25% ethyl alcohol + DMPO (25 mM).

obtained by TBA and ESR methods (Tables 1 and 21, it is indicated that there is a good correlation between both methods. The time courses of signal intensity of DMPO-OH generated during the reaction of H,O* with Cu(II)-tripeptides are shown in Figure 2. Two patterns were observed; one reached maximum at 1 min after mixing components and then decreased sharply, and the other increased gradually with time. The Cu(I1) complexes showing the former pattern are attributable to those of peptides

TABLE 2. Formation of DMPO-OH from the reaction of C&I)-Histidine Complexes with HzO, Copper@

Complexes

Cu(II)_HisGly Cu(II)_GlyHis CU~IINkHiS Cu(II)_HisGlyGly

DMPO-OH 2.5 0.1

Cu(II)_GlyHisGly Cu(II)_GlyGlyHis CdIW-ZisGlyGlyGly CuUI)_GlyGlyHisGly

1.7 2.6 0.1 0.9 3.3 0.6

C&I)_HisHisGlyGly Cu(II)43isGlyHisGly

2.7 0.7

Oligopeptide

ACIIVATION

OF HYDROGEN

Cu(ll)-Tripeptide

PEROXIDE

BY Cu(I1) COMPLEXES

127

Complex

0

Cu(HGG)

0

Cu(GHG)

n

Cu(GGH)

Time (min) FIGURE 2. Dependence of the signal intensity of DMPO-OH on the reaction time.

containing histidyl residue at the N-terminal with high TBARS values, and the latter is those of peptides containing histidyl residue in the second or third position with small TBARS values. The abrupt decrease of signal intensity observed in the Cu(I1) complexes of peptides containing histidine at the Nterminal may be caused by the high reactivities of these complexes against the DMPO-OH adduct formed. The difference of reactivities of C&I) complexes of histidine-containing oligopeptides against H,O, may be attributed to that of the redox potentials of each Cu(I1) ion in the C&I) complexes. Since the redox potentials of Cu(I1) ion are considered to be affected by the coordination structure of C&I) complexes [19], the coordination structures of Cu(I1) complexes of histidine-containing oligopeptides were investigated. In neutral solutions some peptides containing histidyl residue at the N-terminal such as HisGly and HisGlyGly coordinate &th C&I) ion to give dimers [20,21]. For example, it has been reported that Cu(II)-HisGly takes a probable structure of the dimer (Fig. 3a), in which four coordination positions of each Cu(I1) ion are occupied with an amino nitrogen, a deprotonated amide nitrogen, and a carboxylate oxygen of one ligand, and with an imidazole nitrogen of another ligand [211. Similar dimer structure is sug-

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(a)

Prdbable structures of (a) CuWHisGly; (b) CuW-GlyHis; and (cl Cu(II)GlyGlyHis complexes.

FIGURJZ3.

gested in those of Cu(II)-HisHis [22] and Cu(II)-HisHisGlyGly [161. On the other hand, some peptides containing histidyl residue in the second position such as GlyHis (Fig. 3b) and GlyHisGly bind to Cu(I1) ion through an amino nitrogen, a deprotonated amide nitrogen, and an imidazole nitrogen [21l. Further, some peptides containing histidyl residue in the third position such as GlyGlyHis (Fig. 3c), GlyGlyHisGly, and HisGlyHisGly chelate Cu(I1) ion as a quadridentate ligand with four nitrogen donor atoms: that is, amino, two deprotonated amide, and imidazole [21]. From the results mentioned above, it is concluded that Cu(I1) oligopeptide complexes keeping a dimer structure such as Cu(II)_HisGly, -HisHis, -HisGlyGly, and -HisHisGlyGly can easily activate H,O, to yield *OH. 2. Interaction of &(I#-Oligopeptide

Complexes with Superoxide Ion

It.has been established that the HPX/XOD system induces high concentrations of 0; at pH 7.4. This system gave in the presence of DWO a prominent ESR signal of DMPO-0; adduct consisting of twelve lines (aN(l) = 1.43 mT, aH(l) = 1.15 mT, au(l) = 0.13 mT). When Cu,Zn-SOD was added to this O;-genera&g system, the ESR spectrum due to DMPO-0; adduct entirely disappeared. This

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COMPLEXES

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TABLE 3. Abilities of Cu(II)-Hlstidine Oligopeptide Complexes on Dismutation of 0; Copper011 Complexes

% Inhibition of 0; Formation”

Cu,Zn-SOD Cu(II)_HisGly Cu(II)_GlyHis CU(II)_HisHi.5 Cu(II)_HisGlyGly Cu(II)_GlyHisGly Cu(II)_GlyGlyHis Cu(IIMisGlyGlyGly Cu(II)-GlyGlyHisGly CuQI)_HisHisGlyGly CdII~HisGlyHisGly

100 100 loo 100 100 74 100 100 loo 89 90

a Percent inhibition is expressed as follows:

%=

ESR signal intensity of DMPO-0;

in the presence of Cu(I1)

ESR signal intensity of DMPO-0;

in the absence of Cu(I1)

x loo.

indicates that 0; is scavenged by SOD. The reactivities of Cu(I1) complexes with histidine-containing peptides towards 0; were investigated by this 0; scavenging method. The results obtained are summarized in Table 3. It is apparent from Table 3 that all CdII) complexes examined could scavenge 0;. These results suggest that all C&I)-oligopeptide complexes have a SOD-like reactivity towards 0;. These results are in accord with the reports that Cu(II)-HisAla [23], C&I)-HisTyr [23], and Cu(II)-HisGlyGly [24] catalyze 0; dismutation very efficiently. CONCLUSION In the reactions of Cu(I1) complexes of histidine-containing peptides with H,O,, C&I) complexes of peptides containing histidyl residue at the N-terminal could easily activate H,O, to yield -OH as compared with those of peptides containing histidyl residue in the second or third pusition. Further, it is shown that almost all C&I)-oligopeptide complexes have a SOD-like reactivity. I;his work was partially supported by the International Core System for Basic Research (Science and Technob gy Ag ency, Japan). We thank Professor Yoshikxuu Matsushima, Kjor&u College of Phamaam for hti helpfur suggestions.

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