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Clotrimazole Binds to Heme and Enhances Heme-dependent Hemolysis

2002; Elsevier BV; Volume: 277; Issue: 6 Linguagem: Inglês

10.1074/jbc.m107285200

1083-351X

Nguyen Tien Huy, Kaeko Kamei, Takushi Yamamoto, Yoshiro Kondo, Kenji Kanaori, Ryo Takano, Kunihiko Tajima, Saburo Hara,

Mosquito-borne diseases and control

Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: Ka = 6.54 × 108m−2). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S = ½), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite. Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: Ka = 6.54 × 108m−2). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S = ½), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite. chloroquine clotrimazole electron spin resonance histidine-rich protein 2 ferric protoporphyrin IX mesoprotoporphyrin IX Malaria has become a key global threat due to quickly spreading resistance to quinoline-based antimalarial drugs such as quinine, chloroquine (CQ),1 and mefloquine (1Trigg P.I. Kondrachine A.V. Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. ASM press (American Society for Microbiology), Washington, D. C.1998: 11-22Google Scholar). Furthermore, artemisinin-resistant strains ofPlasmodium falciparum have been developed in the laboratory (2Inselburg J. Am. J. Trop. Med. Hyg. 1985; 34: 417-418Crossref PubMed Scopus (37) Google Scholar). Therefore, there has been extensive research into a new series of antimalarial drugs. The antifungal agent clotrimazole (CLT) (Fig.1) inhibits the growth of chloroquine-resistant P. falciparum strains in vitro (3Saliba K.J. Kirk K. Trans. R. Soc. Trop. Med. Hyg. 1998; 92: 666-667Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 4Tiffert T. Ginsburg H. Krugliak M. Elford C. Lew V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 331-336Crossref PubMed Scopus (67) Google Scholar). Mechanisms of the antimalarial activity of CLT have been proposed in relation to Ca2+ ions; CLT inhibits the sarcoplasmic reticulum Ca2+ pump and capacitative Ca2+channels of malaria-infected red blood cells, causing the depletion of intracellular Ca2+ stores (5Benzaquen L.R. Brugnara C. Byers H.R. Gattoni-Celli S. Halperin J.A. Nat. Med. 1995; 1: 534-540Crossref PubMed Scopus (145) Google Scholar, 6Tiffert T. Staines H.M. Ellory J.C. Lew V.L. J. Physiol. 2000; 525: 125-134Crossref PubMed Scopus (39) Google Scholar). Depletion of intracellular Ca2+ induces the activation of protein kinase R and phosphorylation of eukaryotic translation initiation factor 2α, thereby inhibiting protein synthesis in the parasite (7Aktas H. Fluckiger R. Acosta J.A. Salvage J.M. Palakurthi S.S. Halperin J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8280-8285Crossref PubMed Scopus (123) Google Scholar). However, the actual mechanism of CLT antimalarial action at the molecular level remains equivocal. During development and proliferation in human erythrocytes, malaria degrades hemoglobin to use as a major source of amino acids, accompanied by the release of free heme. As free heme is highly toxic to the malarial parasite, the parasite has developed a means of detoxifying heme through polymerization to non-toxic, insoluble hemozoin (8Francis S.E. Sullivan D.J. Goldberg D.E. Annu. Rev. Microbiol. 1997; 51: 97-123Crossref PubMed Scopus (656) Google Scholar) or by degradation with GSH (9Atamna H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 10Ginsburg H. Famin O. Zhang J. Krugliak M. Biochem. Pharmacol. 1998; 56: 1305-1313Crossref PubMed Scopus (269) Google Scholar, 11Platel D.F.N. Mangou F. Tribouley-Duret J. Mol. Biochem. Parasitol. 1999; 98: 215-223Crossref PubMed Scopus (59) Google Scholar), which is found at millimolar concentrations in red blood cells and parasite compartments (12Atamna H. Ginsburg H. Eur. J. Biochem. 1997; 250: 670-679Crossref PubMed Scopus (135) Google Scholar, 13Luersen K. Walter R.D. Muller S. Biochem. J. 2000; 346: 545-552Crossref PubMed Scopus (89) Google Scholar). About 30–50% of free heme is detoxified by polymerization at the trophozoite stage (10Ginsburg H. Famin O. Zhang J. Krugliak M. Biochem. Pharmacol. 1998; 56: 1305-1313Crossref PubMed Scopus (269) Google Scholar, 14Wood P.A. Eton J.W. Am. J. Trop. Med. Hyg. 1993; 48: 465-472Crossref PubMed Scopus (20) Google Scholar, 15Slater A.F.G. Pharmacol. Ther. 1993; 57: 203-235Crossref PubMed Scopus (311) Google Scholar), and the remainder is detoxified by GSH-dependent degradation. The two detoxification processes of free heme are initiated by heme histidine-rich protein (HRP) 2 and heme-GSH complex formation, respectively. Antimalarials such as quinine and CQ also bind to free heme and inhibit its degradation. Furthermore, the imidazole moiety of CLT behaves as an axial ligand, binding free heme. We therefore considered that CLT exerts antimalarial activity by forming complexes with heme, similar to the heme-binding antimalarials, CQ and quinine. In this study, the coordination reaction between CLT and heme was investigated by optical absorption spectroscopy and electron spin resonance (ESR) spectroscopy. The structure of the heme-CLT complex was characterized based on spectroscopic evidence. Furthermore, we compared the effects of CLT and CQ on GSH-dependent heme degradation and heme-induced hemolysis, and we propose a mechanism of antimalarial CLT action. GSH, CLT, CQ, imidazole, and hemin (heme) were from Sigma. Mesoheme was from Porphyrin Products Inc. (Logan, UT). Human blood was drawn from healthy volunteers. Dimethyl sulfoxide (Me2SO) was purchased from Wako Pure Chemicals (Osaka, Japan). All other chemicals were of the highest commercially available grade. At the start of each experiment, a stock heme solution was prepared by dissolving hemin chloride in 20 mm NaOH and then removing the remaining hemin crystals by centrifugation for 10 min at 15,000 rpm. The heme concentration was estimated from absorbance at 385 nm (εmm = 58,400) in 100 mm NaOH (16Shaklai N. Shviro Y. Rabizadeh E. Kirschner-Zilber I. Biochim. Biophys. Acta. 1985; 821: 355-366Crossref PubMed Scopus (91) Google Scholar) and adjusted to 1.0 mm. This stock reagent was stored in the dark on ice and used within 24 h. All absorption spectra were recorded on a Hitachi U-3300 double-beam spectrophotometer (Tokyo, Japan) using a 1.0-cm light-path quartz cuvette at 23 °C. A solution of 17 μm heme in 40% Me2SO and 20 mmHEPES buffer (pH 7.4) revealed Soret at 401 nm and Q band absorption maxima at 493 and 616 nm, and the ratio of absorption of the Soret (401 nm) and Q band (616 nm) was 28.72, indicating that the heme in the present system exists as a monomeric mode (17Beaven G.H. Chen S. D'Albis A. Gratm W.B. Eur. J. Biochem. 1974; 41: 539-546Crossref PubMed Scopus (254) Google Scholar, 18Collier G.S. De Pratt J.M. Wet R. Tshabalala C.F. Biochem. J. 1979; 179: 281-289Crossref PubMed Scopus (60) Google Scholar, 19Egan T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar). The optical absorption spectra of the heme-CLT and heme-imidazole complexes were recorded 5 min after adding CLT (final concentration, 100 μm) and imidazole (final concentration, 500 μm) to heme (final concentration, 17 μm) in 40% Me2SO buffered with 20 mm HEPES (pH 7.4). The total volume of the reaction mixture was 1.0 ml. Differential absorption spectra were measured on a Hitachi U-3300 spectrophotometer as follows. The drug, CLT or CQ, was added sequentially to a sample cuvette containing heme solution. The reference compartment held two cuvettes, one containing an identical heme solution aliquot to which a buffer other than the drug was added and the other containing a solution without heme and the same amount of the drug. In the case of CLT titration, both the sample cuvette and the first reference cuvette contained 17 μm heme in 40% Me2SO buffered by 20 mm HEPES (pH 7.4), and the second reference cuvette contained the same solution without heme. Increasing amounts of CLT (0 μm–105.6 μmin 6.6 μm increments) in Me2SO were titrated with the contents of the sample cuvette and the second reference cuvette, in which the total volume of the reaction mixture was maintained at 1.0 ml. Before adding CQ, the sample cuvette and the first reference cuvette contained 5 μm heme in 40% Me2SO buffered by 20 mm HEPES (pH 7.4), and the second reference cuvette contained the same solution without heme. During the titration of heme-CQ complex formation, increasing amounts of CQ (0–36 μm in 4 μm increments) were added to both the sample and the second reference cuvettes, where the total volume of the reaction mixture was 1.0 ml during the titration. All differential spectra were recorded at wavelengths between 350 and 700 nm, and the concentrations of heme-CLT and heme-CQ complexes were evaluated based on absorbance at 416 and 401 nm, respectively. The binding mode of CLT and CQ to heme was analyzed in terms of Hill (20Van Holde K.E. Physical Biochemistry. Prentice Hall, Englewood Cliffs, NJ1971: 62-64Google Scholar, 21Brault D. Rougee M. Biochem. Biophys. Res. Commun. 1974; 57: 654-659Crossref PubMed Scopus (118) Google Scholar) and Scatchard (22Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17789) Google Scholar, 23Cantor C.R. Schimmel P.R. Biophysical Chemistry, Part III. W. H. Freeman, San Francisco, CA1980: 849-863Google Scholar) plots. The equilibrium association constants for the formation of heme-CLT and heme-CQ complexes, as well as the number of ligands that bind to heme, were calculated from Hill plots using Eq. 1,H+nL⇄H(L)nEquation 1 and analyzed using the standard equation (Eq. 2),logA−A0A∞−A=logKa+nlog[L]Equation 2 where A0, A∞, and A are the absorbance of the initial, final, and mixed species, respectively; H represents heme; L is the ligand (CLT or CQ); n is the number of ligand molecules that bind to heme; and Ka is the equilibrium association constant of the heme-ligand complex. ESR measurements were continued for heme-CLT complexes that were formed in Me2SO at molar ratios of heme to CLT of 1:1, 1:1.5, 1:2, 1:4, and 1:8. After an overnight incubation at room temperature, the ESR spectrum of heme-CLT complex was recorded at 4.2 K by a JES-TE 300 spectrometer with 100-kHz field modulation. The integrated frequency counter monitored the microwave frequency of each measurement. The magnetic field strength was calibrated by hyperfine splitting of Mn(II) ion (8.69 milliteslas (mT)) doped in MgO powder. Powdered lithium-tetracyanoquinodimethane radical (g = 2.0025) was used as the standard g value. The ESR data were analyzed and calibrated using a Winrad system (Radical Research Inc., Tokyo). The typical conditions for ESR measurements were as follows: microwave power, 6.0 milliwatts; modulation magnitude, 0.68 mT; sweep range 30 mT to 500 mT; sweep time, 4 min; and time constants, 0.1 s. Heme degradation by GSH was monitored by measuring spectral change as described by Atamna and Ginsburg (9Atamna H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Fresh GSH stock solution (200 mm) was prepared in isotonic standard buffer (50 mm sodium phosphate containing 68 mmNaCl, 4.8 mm KCl, and 1.2 mm MgSO4, pH 7.4) (24Chou A.C. Chevli R. Fitch C.D. Biochemistry. 1980; 19: 1543-1549Crossref PubMed Scopus (325) Google Scholar, 25Fitch C.D. Chevli R. Gonzalex Y. Antimicrob. Agents Chemother. 1974; 6: 757-762Crossref PubMed Scopus (27) Google Scholar). Heme (final concentration, 3 μm) and GSH (final concentration, 2 mm) were mixed in isotonic standard buffer (pH 7.4) and incubated at 37 °C. Absorption spectra (300–600 nm) were recorded at 6-min intervals after mixing, using the same spectrophotometer. The rate constant and t½of GSH-dependent heme degradation in the absence of CLT and CQ were calculated from the decrease of absorbance at 365 nm in terms of first-order reaction kinetics. In the presence of CLT (6 μm) or CQ (6 μm), the time-dependent spectral measurements were obtained by the same procedure. CLT in Me2SO and CQ in HEPES buffer (200 mm, pH 7.4) were all prepared as 3 mmstock solutions. Heme (3 μm), GSH (2 mm), and either CLT or CQ (6 μm) were mixed in 0.2 mHEPES buffer (pH 7.4) and incubated at 37 °C. In the control experiment, Me2SO (final concentration, 0.2% (v/v)) was added to the mixture of heme (3 μm) and GSH (2 mm) instead of CLT and CQ. The time-dependent change of absorbance at 396 nm was recorded as an indicator of heme degradation. Fresh blood from healthy donors was heparinized (1 mg of heparin/ml blood) to suppress clotting. The erythrocytes were separated from plasma by centrifugation at 1,500 × gfor 3 min and washed six times with isotonic standard buffer. Thereafter, the effects of CLT and CQ on hemolysis induced by heme were examined in 0.5% cell suspensions in isotonic standard buffer. Erythrocyte suspensions (0.6 ml) were shaken with various concentrations of heme (0–20 μm) and CLT or CQ (0, 1, 5, and 10 μm) at 37 °C for 150 min at 140 cycles/min. Intact erythrocytes were then removed by centrifugation at 1,500 × g for 3 min, and the amount of hemoglobin released from the hemolyzed erythrocytes into the supernatant was determined by measuring absorbance at 578 nm (26Shviro Y. Shaklai N. Biochem. Pharmacol. 1987; 36: 3801-3807Crossref PubMed Scopus (51) Google Scholar). After the pelleted intact erythrocytes were lysed with water and centrifuged to obtain the supernatant, the hemoglobin content in intact erythrocytes was measured as absorbance at 578 nm. The degree of hemolysis was calculated from the ratio of hemoglobin content released from erythrocytes hemolyzed by heme to the total heme content of the erythrocytes (26Shviro Y. Shaklai N. Biochem. Pharmacol. 1987; 36: 3801-3807Crossref PubMed Scopus (51) Google Scholar). When using heme-bound erythrocytes, 0.5% of red blood cells in isotonic standard buffer, pH 7.4, were incubated with 10 μm heme at room temperature for 10 min. The erythrocyte suspension was separated by centrifugation at 1,500 ×g for 3 min, and the pellet was washed three times with isotonic standard buffer to remove free heme, thus providing heme-bound erythrocytes. A sample of 0.6 ml of a 0.5% suspension of heme-bound erythrocytes was prepared in isotonic standard buffer, Me2SO (1%), CLT (10 μm), CQ (10 μm), or GSH (2.5 mm) was then added, and the mixture was incubated at 37 °C for 2 h. The hemolysis degree was calculated from three such experiments. Fig.2, curve 1, shows Soret and Q band absorption at 401, 493, and 616 nm by heme (17 μm) in 40% Me2SO, which is characteristic of high spin ferric complexes assuming a five-coordinate structure (27Kaminsky L.S. Byrne M. Davison A.J. Arch. Biochem. Biophys. 1972; 150: 355-361Crossref PubMed Scopus (29) Google Scholar,28Pasternack R.F. Gillies B.S. Stahlbush J.R. J. Am. Chem. Soc. 1978; 100: 2613-2619Crossref Scopus (50) Google Scholar) with weak axial ligand such as water or chloride anion. When excess CLT (final concentration, 100 μm) was added to the mixture, the Soret shifted toward red wavelengths at 412 nm, and Q band absorption was evident at 536 and 560 nm, as shown in Fig. 2,curve 2. The observed spectrum was classified into a six-coordinate ferric complex having nitrogenous ligands at both axial positions. In fact, the spectroscopic properties coincided with those of similar solutions of heme and imidazole (Soret, 410 nm; Q bands, 535 and 560 nm) shown in Fig. 2, curve 3. Furthermore, heme-bis-imidazole complexes have similar spectra (29Babcock G.T. Widger W.R. Cramer W.A. Oerling W.A. Metz J.G. Biochemistry. 1985; 24: 3638-3645Crossref PubMed Scopus (174) Google Scholar, 30Katagiri M. Tsutsui K. Yamano T. Shimonishi Y. Ishibashi F. Biochem. Biophys. Res. Commun. 1987; 149: 1070-1076Crossref PubMed Scopus (25) Google Scholar), as summarized in Table I. These results support the notion that CLT, like imidazole, has affinity for the heme chromophore. It is likely that the imidazole moiety of CLT is the nitrogenous donor.Table IAbsorption maxima for protoheme (ferric protoporphyrin IX) complexed with CLT, CQ, imidazole, N-methylimidazole, or hexapeptide containing two histidine residuesSampleAxial ligandAdditiveSolventAbsorptionReferencenmProtohemenonenone40% Me2SO1-a40% Me2SO, 20 mm HEPES buffer at pH 7.4.401493616This workProtohemeCLTnone40% Me2SO1-a40% Me2SO, 20 mm HEPES buffer at pH 7.4.412536560This workProtohemeImidazolenone40% Me2SO1-a40% Me2SO, 20 mm HEPES buffer at pH 7.4.410535560This workProtohemeCLTnoneHEPES buffer1-b20 mm HEPES buffer, pH 7.4.416533564This workProtohemeCLTGSHHEPES buffer1-b20 mm HEPES buffer, pH 7.4.416533564This workProtohemenoneCQHEPES buffer1-b20 mm HEPES buffer, pH 7.4.390594This workProtohemenoneCQ + GSHHEPES buffer1-b20 mm HEPES buffer, pH 7.4.390594This workProtohemeN-methylimidazolenonePhosphate buffer1-cpH 7.4.413535564Ref.29Babcock G.T. Widger W.R. Cramer W.A. Oerling W.A. Metz J.G. Biochemistry. 1985; 24: 3638-3645Crossref PubMed Scopus (174) Google ScholarProtohemeHexapeptidenonePhosphate buffer1-dpH 7.6.415538566Ref.30Katagiri M. Tsutsui K. Yamano T. Shimonishi Y. Ishibashi F. Biochem. Biophys. Res. Commun. 1987; 149: 1070-1076Crossref PubMed Scopus (25) Google Scholar1-a 40% Me2SO, 20 mm HEPES buffer at pH 7.4.1-b 20 mm HEPES buffer, pH 7.4.1-c pH 7.4.1-d pH 7.6. Open table in a new tab The heme-CLT complex in the absence of Me2SO gave a similar absorption spectrum, as indicated by a Soret band at 416 nm and Q bands at 533 and 564 nm (Table I). In contrast, the spectrum of the heme-imidazole complex in the absence of Me2SO included a broad peak at 433 nm (data not shown), which is derived mainly from the aggregated form of heme (31Gallagher W.A. Elliotte W.B. Biochem. J. 1968; 108: 131-136Crossref PubMed Scopus (28) Google Scholar, 32Campbell V.M. Studies on Hemin and Cobalt Corrinoids in Aqueous SolutionPh.D. thesis. University of Witwaterstrand, 1981Google Scholar). The heme-CLT complex, prepared in HEPES buffer in the absence of Me2SO, was quite stable under ambient conditions. The values of the absorption maxima of the heme-CLT complex in Me2SO and in HEPES buffer did not vary significantly, and heme precipitation was undetectable even in HEPES buffer. These results provide potent evidence of the ability of CLT to stabilize the monomeric form of heme and to inhibit the formation of polymeric heme in aqueous solution. To characterize the binding of heme with CLT, spectrophotometric heme titration was performed by measuring the differential spectra between heme and heme-CLT complex at various CLT concentrations. As described under "Experimental Procedures," aqueous-Me2SO (40% v/v) buffered by 20 mm HEPES buffer, pH 7.4, was used because heme in this solution should form a monomer at concentrations up to 26.6 μm (17Beaven G.H. Chen S. D'Albis A. Gratm W.B. Eur. J. Biochem. 1974; 41: 539-546Crossref PubMed Scopus (254) Google Scholar, 18Collier G.S. De Pratt J.M. Wet R. Tshabalala C.F. Biochem. J. 1979; 179: 281-289Crossref PubMed Scopus (60) Google Scholar, 19Egan T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar). Fig.3A shows that CLT perturbs the spectrum of heme, indicating interaction between the drug and heme. Continuous addition of CLT into a heme solution achieves conversion of the heme spectrum to a form with lower Soret molar absorption and a Soret maximum shifted to a longer wavelength. The absorption spectra changed as the CLT concentration increased, through isosbestic points at 409, 467, 514, and 585 nm, indicating that only two absorbing species are present in the reaction mixture. During heme-CQ complex formation, the spectrum change was accompanied by a significant decrease in the intensity of monomeric heme at the Soret and Q bands (Fig. 3B), as described by Egan et al. (19Egan T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar), indicating interaction between CQ and heme. The effects of heme interactions with CLT and CQ on titration behavior were analyzed using Hill plots (20Van Holde K.E. Physical Biochemistry. Prentice Hall, Englewood Cliffs, NJ1971: 62-64Google Scholar, 21Brault D. Rougee M. Biochem. Biophys. Res. Commun. 1974; 57: 654-659Crossref PubMed Scopus (118) Google Scholar) to determine the number of molecules bound to heme in aqueous Me2SO. Hill plots of our binding data in Fig. 4 show heme-CLT complexes at 17 μm heme and heme-CQ complexes at 5 μm heme. The slopes of these linear graphs are 2 and 1, respectively, within experimental error. The same data are presented in Scatchard plots in Fig.5 (22Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17789) Google Scholar, 23Cantor C.R. Schimmel P.R. Biophysical Chemistry, Part III. W. H. Freeman, San Francisco, CA1980: 849-863Google Scholar). The straight lineindicates the absence of cooperative interaction between heme and CQ, whereas a curved graph is observed for heme-CLT complex, indicating cooperation and the involvement of non-identical interacting binding sites. The analysis using the Hill plot demonstrates that heme binds two CLT molecules with an association constant (Ka) of 6.54 × 108m−2, whereas heme binds one molecule of CQ with a Ka of 1.71 × 105m−1 in aqueous 40% Me2SO at pH 7.4. We further clarified the electronic and coordination structures of the heme-CLT complex by ESR spectroscopy. We recorded ESR spectra at 4.2 K for heme-CLT complexes prepared in Me2SO, as described under "Experimental Procedures." Before adding CLT, the observed ESR spectrum (Fig. 6, curve 1) of heme (0.5 mm) contained the line and g values (g = 6 and g = 2.0) typical of the ferric high spin (S =52) species, taking five-coordinate geometry into account (33Momenteau M. Biochim. Biophys. Acta. 1973; 304: 814-827Crossref PubMed Scopus (62) Google Scholar). A weak signal is always observed at about g = 4.3, which may be due to non-heme iron from decomposed heme (34Tajima K. Inorg. Chim. Acta. 1989; 163: 115-122Crossref Scopus (34) Google Scholar) or impurities in the sample tube and Dewar assembly. On adding CLT (final concentration, 100 mm) to the reaction mixture, the ESR signal intensity of the high spin species decreased significantly with the concomitant formation of a new paramagnetic species with a distorted rhombic ESR line (Fig. 6, curves 2 and 3; g1 = 1.46, g2 = 2.26, and g3 = 2.98), which was characteristic of ferric low spin complex (S = ½) having strong axial ligands at both axial positions. Upon the further addition of CLT, the molar ratio of CLT/heme reached 8, and the ESR signal of the new species was recorded exclusively, suggesting that CLT tended to shift the equilibrium to form the low spin ferric species (data not shown). The observed ESR shape of the low spin species line was quite similar to that recorded for a frozen mixture of heme-imidazole complex (data not shown). In addition, the observed g values of those complexes agreed well (e.g. protoheme-imidazole and protoheme-hexapeptide complex) (TableII). This provided experimental evidence that both complexes, heme-CLT and heme-imidazole complexes, have similar ligands at the axial positions.Table IIThe g values and crystal field parameters (calculated by using Bohan's proposal (37Bohan T.L. J. Magn. Reson. 1977; 26: 109-118Google Scholar)) of heme-CLT complex and relating ferric low-spin complexesSampleg1g2g3‖R/μ‖‖μ/λ‖ReferenceFe3+ mesoprotoporphyrin IX (CLT)22-a100% Me2SO.1.462.262.980.5663.121This workFe3+ mesoprotoporphyrin IX (imidazole)22-bPhosphate buffer, pH 7.4.1.482.242.980.5363.316This workCytochrome b-559 (from maize)2-bPhosphate buffer, pH 7.4.1.542.272.940.5703.364Ref.29Babcock G.T. Widger W.R. Cramer W.A. Oerling W.A. Metz J.G. Biochemistry. 1985; 24: 3638-3645Crossref PubMed Scopus (174) Google ScholarFe3+ protoporphyrin IX (N-methylimidazole)22-bPhosphate buffer, pH 7.4.1.522.262.950.5603.342Ref.29Babcock G.T. Widger W.R. Cramer W.A. Oerling W.A. Metz J.G. Biochemistry. 1985; 24: 3638-3645Crossref PubMed Scopus (174) Google ScholarClorodeuterohemin dimethyl ester (imidazole)22-cChloroform.1.532.252.920.5523.592Ref.33Momenteau M. Biochim. Biophys. Acta. 1973; 304: 814-827Crossref PubMed Scopus (62) Google Scholar2-a 100% Me2SO.2-b Phosphate buffer, pH 7.4.2-c Chloroform. Open table in a new tab The absorption spectrum of heme in HEPES buffer (200 mm, pH 7.4) exhibited two Soret bands at 385 and 342 nm, representing the monomer and dimer form, respectively (Fig.7) (35Sahini V.E. Dumitrescu M. Volanschi E. Birla L. Diaconu C. Biophys. Chem. 1996; 58: 245-253Crossref PubMed Scopus (16) Google Scholar), whereas the spectrum of heme in 40% Me2SO revealed only one Soret band at 401 nm, indicating the monomer form of heme (Fig. 2) (17Beaven G.H. Chen S. D'Albis A. Gratm W.B. Eur. J. Biochem. 1974; 41: 539-546Crossref PubMed Scopus (254) Google Scholar, 18Collier G.S. De Pratt J.M. Wet R. Tshabalala C.F. Biochem. J. 1979; 179: 281-289Crossref PubMed Scopus (60) Google Scholar, 19Egan T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar). The maximal absorption of the Soret of heme (3 μm) was shifted to 365 nm after adding GSH (2 mm) as described in Ref. 9Atamna H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, probably due to the formation of GSH-heme complex. Fig. 7 shows that the Soret absorption of heme complexed with GSH declined rapidly as described in Ref. 9Atamna H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, indicating the degradation of heme by GSH. The rate constant and t½ of the heme degradation were calculated from fitting to the first-order reaction as 4.5 × 10−4 s−1 and 1,540 s, respectively, in isotonic standard buffer at pH 7.4. The absorption spectra of heme-CLT and of heme-CQ complexes did not change upon the addition of GSH, indicating that neither complex interacted with GSH (data not shown). The GSH-dependent degradation of heme (3 μm) in the presence of CLT (6 μm) or CQ (6 μm) was monitored as a decrease of absorbance at 396 nm because heme-CLT complex and heme-CQ complex have similar molecular absorption coefficients at 396 nm. The results shown in Fig. 8 indicate that CLT and CQ inhibit GSH-dependent heme degradation. Hemolysis experiments were performed using fresh blood cells as described under "Experimental Procedures." Up to only 2.5% hemolysis occurred in controls in which no heme was added. The hemolysis induced by the presence of heme was potentiated by CLT as well as by CQ (Fig. 9A), and the effects depended on the concentrations of both heme and the added agents. We also observed that CLT alone at up to 20 μmhad no effect on hemolysis in the absence of heme (data not shown). Therefore, the enhancement of heme-dependent hemolysis of erythrocytes may be caused by the formation of heme-CLT complex. The amount of heme-dependent hemolysis enhanced by CQ was almost identical to that observed in a previous study (36Dutta P. Fitch C.D. J. Pharmacol. Exp. Ther. 1983; 225: 729-734PubMed Google Scholar). The similar enhancement of heme-dependent hemolysis by CLT and CQ indicates that they have the same potential at high concentrations of heme (5–20 μm), as shown in Fig.9A. However, at lower heme