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Ampicillin serves as an electron donor
Inr. J. Biochem.Vol. 22, No. Il. pp. 1291-1293, 1990 Printed in Great Britain. All rights -cd
AMPICILLIN
0020-711X/90 53.00 + 0.00 Copyright @ 1990 Pergamon Press plc
SERVES AS AN ELECTRON SHIOEN~BU
Division
of
Respiratory
DONOR
UmKI
Diseases, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-01, Japan [Fax 0864-62-I 1991 (Received 19 February 1990)
Abatraet-1. The effect of ampicillin on cytochrome c reduction and on the superoxide production of human neutrophils stimulated by phorbol myristate acetate (PMA) was investigated. 2. Ampicillin did not stimulate the superoxide production of intact (resting) neutrophils and not amplify the superoxide production of neutrophils stimulated by phorbol mytistate acetate (PMA). 3. However, ampicillin dose-dependently increased the reduction of cytochrome c. 4. In addition, 50 mM ampicillin stimulated a superoxide dismutase-inhibitable reduction of cytochrome c by 0.70 + 0.02 (mean f SD) nmol/min and a superoxide dismutase-noninhibitable reduction of cytochrome c by 2.08 f 0.03 (mean f SD) nmol/min. 5. These results suggest that ampicillin serves as an electron donor and/or a superoxide generator.
INTRODUCI1ON Some antibiotics have been shown to have biochemical actions other than simple antimicrobial activity. For instance, they affect the function of polymorphonuclear leukocytes (neutrophils) (Hauser and Remington, 1982; Mandell, 1982). Concerning antimicrobial agents, not only the susceptibility of the bacteria but also any possible positive or negative effects of the antimicrobial agent on the host defence system should be investigated. Neutrophils play a crucial role in the host defence system (Stossel, 1974) and kill bacteria in various ways involving activation of their oxidative metabolism (Klebanoff, 1975). Activation of the neutrophil oxidative metabolism (respiratory burst) is characterized by strong cyanide-insensitive oxygen consumption (Sbarra and Kamovsky, 1959) and by concomitant production of 0; and H,O1 (Babior, 1978). Superoxide is primarily produced through the activation of plasma membrane-bound NADPH oxidase (EC 1.6.99.6) by stimulation with chemoattractants (Rossi et al., 1972). Several investigators have reported effects of antimicrobial agents on chemiluminescence of neutrophils stimulated by various chemoattractants (Mandell, 1982; Briheim and Dahlgren, 1987; Duncker and Ullmann, 1986). However, little information about effects of antimicrobial agents on the respiratory burst of neutrophils and cytochrome c reduction involved with an electron transport system is available. In the present study, the effect of ampicillin on the superoxide production of human neutrophils stimulated by phorbol my&ate acetate (PMA) and on cytochrome c reduction involved with an electron transport system. MATERIALS AND METHODS The following chemicals were obtained from commercial sources: bovine erythrocyte superoxide dismutase, cytochrome c (type III), /?-NADPH (type I), PMA, dextran (average M, 78,000) and sodium a-aminobenzyl penicillin
(ampicillin) (Sigma Chemical Co., St Louis, MO.); Dulbecco’s calcium and magnesium-free phosphate buffered saline and Hanks balanced salt solution (HBSS) (Gibco Laboratories, Grand Island, N.Y.); Ficoll-Paque (Pharmacia P-L Bio-chemicals, Piscataway, N.J.). Other chemicals were of the highest purity available from commercial sources. Human neutrophils of >98% purity were prepared as previously described (McPhail et al., 1985; Umeki, 1990) using acid citrate dextrose as the anticoagulant, dextran to sediment erythrocytes, and Ficoll-Paque to separate mononuclear cells from neutrophils. Superoxide production by intact neutrophils stimulated by PMA was measured by a discontinuous assay in which superoxide dismutastinhibitable reduction of cytochrome c was followed by scanning between 530 and 570nm, as previously described (Umeki and Soejima, 1990). Neutrophils (2 x 10J cell eq/cuvette) were incubated in an assay HESS medium containing 0.12 mM cytochrome c and the desired concentrations of ampicillin for 2 min at 37°C before the reactions were initiated by adding PMA (0.3 pg/cuvette). Assay mixtures were incubated for 4 min at 37”C, in a total volume of 1.Oml. The reference cuvette also received 20 pg of superoxide dismutase. Assay mixtures for the cytochrome c reduction due to ampicillin contained 0.12 mM cytochrome c and the desired concentrations of ampicillin, with alterations as noted in the table and figure legends, in an assay HBSS medium. Superoxide production and cytochrome c reduction were calculated using an extinction coefficient of EE, = 19.6 mM-‘/cm (Yonetani, 1965). The protein concentration was determined by the method of Smith er ol. (1985), using the bicinchoninic acid protein assay reagent from Pierce Chemical Co. (Rockford, Ill.). Protein concentration of neutrophil suspension was 860 * 23 (mean f SD) pg protein/IO’ cell eq. RESULTS
Table 1 shows effects of ampicillin on the reduction of cytochrome c and on the superoxide generation of human neutrophils stimulated by PMA. Intact cells produced a superoxide dismutase-inhibitable reduction of cytochrome c by 0.09 + 0.02 (mean + SD) nmol/min/2 x IO5cell eq and a superoxide dismutasenoninhibitable reduction of cytochrome c by 0.06 f 0.02 (mean f SD) nmol/min/2 x IO’ cell eq.
1291
1292 Table
1.Effects of
ampicillin on cytochrome c reduction
cuvct1c
______-.
Refere”LX
Cytochrome c reduction (nmol/min)
Sampk
SOD SOD SOD SOD SOD SOD SOD SOD SOD SOD/PMA SOD/ABPC SOD/cells SOD/oclls/PMA SOD/alls/ABPC SOD/cells/PMA/ABPC
Mean f SD 0.03 * 0.01 2.08 + 0.03 0.06 f 0.02 0.03 f 0.01 2.72 f 0.04 0.16 f 0.03 2.83 f 0.04 2.92 f 0.05 5.84 f 0.04 0.03 * 0.01 0.70 2 0.02 0.09 f 0.02 2.88 f 0.03 0.75 f 0.04 3.76 + 0.05
SOD/PMA SOD/ABPC SOD/LXllS PMA ABPC Cells Cclls/PMA Cells/ABPC Cclls/PMA/ABPC PMA ABPC Cells Cells/PMA Cclls/ABPC Cells/PMA/ABPC
The assay method is described in Materials and Methods. All cuvct~cs contained HBSS buffer and cytochromc c. The results show” are mean f SD of thra different experiment. SOD, superoxide dismutasc.
PMA did not produce any significant changes in both superoxide dismutase-inhibitable and noninhibitable reduction of cytochrome c. However, neutrophils stimulated by 0.3 pg of PMA produced superoxide by 2.88 f 0.03 (mean f SD) nmol/min/2 x 10’ cell eq. On the other hand, 50 mM ampicillin amplified a superoxide dismutase-inhibitable reduction of cytochrome c by 0.70 + 0.02 (mean * SD) nmol/min/ 2 x 10’ cell eq and a superoxide dismutase-noninhibitable reduction of cytochrome c by 2.08 + 0.03 (mean f SD) nmol/min/2 x lo5 cell eq. However, ampicillin did not stimulate the superoxide production of resting cells and not amplify the superoxide production of neutrophils stimulated by PMA.
0
to
20
30
Concentration
40
50
of tmpici
60
I I in
TO
60
(mhl)
Fig. I. Dose-dependent effects of ampicillin on the reduction of cytochromc c. The assay method is described in Materials and Methods. The results shown are the mean f SD of three different
experiments.
Figure 1 shows dose-dependent effect of ampicillin on the reduction of cytochrome c which is superoxide dismutase-inhibitable and noninhibitable. Cytochrome c reduction increased in a linear fashion as an increasing concentration of ampicillin. DISCL’SSION
agents influence the function of phagocytic neutrophils (Hauser and Remington, 1982; Mandell, 1982). Briheim and Dahlgren (1987) demonstrated an inhibitory effect of penicillin G and ampicillin on luminol-enhanced chemiluminescence of neutrophils stimulated by a chemoattractant formylmethionyl-leucyl-phenylalanine. However, Duncker and Ullmann (1986) reported that ampicillin does not influence luminolenhanced chemiluminescence reaction of neutrophils stimulated by an opsonized zymosan. In the present study, the results obtained indicated no significant effects of ampicillin on the superoxide production of neutrophils stimulated by PMA (respiratory burst phenomenon). Ampicillin (Fig. 2) has been shown to be an antibiotic having microbicidal activity against both Gram-positive and negative bacteria (May and Delves, 1965). The results obtained here indicated that ampicillin may amplify both superoxide dismutase-inhibitable and noninhibitable reduction of cytochrome c, suggesting that this drug serves as an electron donor, like NAD(P)H and ascorbic acid, supplying superoxide dismutase or cytochrome c with an electron. The recent investigation by Scarpa et al. (1983) has demonstrated that one molecule of superoxide radical is generated for each molecule of ascorbic acid oxidized. Based on the evidence, it is considered that ampicillin, like ascorbic acid, generates the superoxide when it is oxidized. It is concluded that ampicillin may serve as an electron donor and/or a superoxide generator. It
is well known that antimicrobial
Acknowledgements-l J.T. Cumutte
Fig. 2. Structure of ampicillin (sodium a-aminobenzylpenicillin).
wish to thank Drs B.M. Babior and of the Research lnstitute of Scripps Clinic, La Jolla of the United States, for the encouragement given me lo undertake these studies and for their critical advice and review.
Ampicillin is an electron donor REFERENCES Babior B. M. (1978) Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298, 659-668. Briheim G. and Dahlgren C. (1987) Influence of antibiotics on formylmethionyl-leucyl-phenylalanine-induced leukocyte chemiluminescence. Antimicrob. Agents Chemother. 31, 763-767.
Duncker D. and Ullmann U. (1986) Influence of various antimicrobial agents on the chemiluminescence of phagocytosing human granulocytes. Chemotherapy 32, 18-24. Hauser W. E. Jr and Remington J. S. (1982) Effect of antibiotics on the immune response. Am. J. Med. 72, 71 I-716. Klebanoff S. J. (1975) Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hemarol. 12, 117-142.
Mandell L. A. (1982) Effect of antimicrobial and antineoplastic drugs on the phagocyte and microbicidal function of the polymorphonuclear leukocyte. Rm. infecr. Dis. 4, 683697. May J. R. and Delves D. M. (1965) Treatment of chronic bronchitis with ampicillin. Lancer 1, 929-933. McPhail L. C., Shirley P. S., Clayton C. C. and Snyderman R. (1985) Activation of the respiratory burst enzyme system from human neutrophils in a cell-free system. Evidence for a soluble cofactor. J. clin. Inuesr. 75, 1735-1739. Rossi F., Romeo D. and Patriarca P. (1972) Mechanism of phagocytosis-associated oxidative metabolism in poly-
1293
morphonuclear leucocytes and macrophages. J. rericuloendothel. Sot. 12, 127-149. Sbarra A. J. and Karnovsky M. L. (1959) The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J. biol. Chem. 234, 1355-I 362. Scarpa M.. Stevanato R., Viglino P. and Rigo A. (1983) Superoxide ion as active intermediate in the autoxidation of ascorbate by molecular oxygen: effect of superoxide dismutase. J. biol. Chem. 258, 6695497. Smith P. K., Krohn R. I., Hermanson G. T.. Mallia A. K.. Gartner F. H., Provenzano M. D., Fujimoto E. K.. Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyr. Eiochem.
150. 76-85.
Stossel T. P. (1974) Phagccytosis. N. Engl. J. Med. 290, 7 I7--723, 774 -780, 833-839. Umeki S. (1990) Human neutrophil cytosolic activation factor of the NADPH oxidase: characterization of activation kinetics. J. biol. Chem. 265, 5049-5054. Umeki S. and Soejima R. (1990) Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems. Biochim. biophys. Acra 1052, 21 I 215.
Yonetani T. (1965) Studies on cytochrome c peroxidase. II. Stoichiometry between enzyme, H,H,, and ferrocytochrome c and enzymic determination of extinction coefficients of cytochrome c. J. biol. Chem. 240, 4509 4514.
AMPICILLIN
0020-711X/90 53.00 + 0.00 Copyright @ 1990 Pergamon Press plc
SERVES AS AN ELECTRON SHIOEN~BU
Division
of
Respiratory
DONOR
UmKI
Diseases, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-01, Japan [Fax 0864-62-I 1991 (Received 19 February 1990)
Abatraet-1. The effect of ampicillin on cytochrome c reduction and on the superoxide production of human neutrophils stimulated by phorbol myristate acetate (PMA) was investigated. 2. Ampicillin did not stimulate the superoxide production of intact (resting) neutrophils and not amplify the superoxide production of neutrophils stimulated by phorbol mytistate acetate (PMA). 3. However, ampicillin dose-dependently increased the reduction of cytochrome c. 4. In addition, 50 mM ampicillin stimulated a superoxide dismutase-inhibitable reduction of cytochrome c by 0.70 + 0.02 (mean f SD) nmol/min and a superoxide dismutase-noninhibitable reduction of cytochrome c by 2.08 f 0.03 (mean f SD) nmol/min. 5. These results suggest that ampicillin serves as an electron donor and/or a superoxide generator.
INTRODUCI1ON Some antibiotics have been shown to have biochemical actions other than simple antimicrobial activity. For instance, they affect the function of polymorphonuclear leukocytes (neutrophils) (Hauser and Remington, 1982; Mandell, 1982). Concerning antimicrobial agents, not only the susceptibility of the bacteria but also any possible positive or negative effects of the antimicrobial agent on the host defence system should be investigated. Neutrophils play a crucial role in the host defence system (Stossel, 1974) and kill bacteria in various ways involving activation of their oxidative metabolism (Klebanoff, 1975). Activation of the neutrophil oxidative metabolism (respiratory burst) is characterized by strong cyanide-insensitive oxygen consumption (Sbarra and Kamovsky, 1959) and by concomitant production of 0; and H,O1 (Babior, 1978). Superoxide is primarily produced through the activation of plasma membrane-bound NADPH oxidase (EC 1.6.99.6) by stimulation with chemoattractants (Rossi et al., 1972). Several investigators have reported effects of antimicrobial agents on chemiluminescence of neutrophils stimulated by various chemoattractants (Mandell, 1982; Briheim and Dahlgren, 1987; Duncker and Ullmann, 1986). However, little information about effects of antimicrobial agents on the respiratory burst of neutrophils and cytochrome c reduction involved with an electron transport system is available. In the present study, the effect of ampicillin on the superoxide production of human neutrophils stimulated by phorbol my&ate acetate (PMA) and on cytochrome c reduction involved with an electron transport system. MATERIALS AND METHODS The following chemicals were obtained from commercial sources: bovine erythrocyte superoxide dismutase, cytochrome c (type III), /?-NADPH (type I), PMA, dextran (average M, 78,000) and sodium a-aminobenzyl penicillin
(ampicillin) (Sigma Chemical Co., St Louis, MO.); Dulbecco’s calcium and magnesium-free phosphate buffered saline and Hanks balanced salt solution (HBSS) (Gibco Laboratories, Grand Island, N.Y.); Ficoll-Paque (Pharmacia P-L Bio-chemicals, Piscataway, N.J.). Other chemicals were of the highest purity available from commercial sources. Human neutrophils of >98% purity were prepared as previously described (McPhail et al., 1985; Umeki, 1990) using acid citrate dextrose as the anticoagulant, dextran to sediment erythrocytes, and Ficoll-Paque to separate mononuclear cells from neutrophils. Superoxide production by intact neutrophils stimulated by PMA was measured by a discontinuous assay in which superoxide dismutastinhibitable reduction of cytochrome c was followed by scanning between 530 and 570nm, as previously described (Umeki and Soejima, 1990). Neutrophils (2 x 10J cell eq/cuvette) were incubated in an assay HESS medium containing 0.12 mM cytochrome c and the desired concentrations of ampicillin for 2 min at 37°C before the reactions were initiated by adding PMA (0.3 pg/cuvette). Assay mixtures were incubated for 4 min at 37”C, in a total volume of 1.Oml. The reference cuvette also received 20 pg of superoxide dismutase. Assay mixtures for the cytochrome c reduction due to ampicillin contained 0.12 mM cytochrome c and the desired concentrations of ampicillin, with alterations as noted in the table and figure legends, in an assay HBSS medium. Superoxide production and cytochrome c reduction were calculated using an extinction coefficient of EE, = 19.6 mM-‘/cm (Yonetani, 1965). The protein concentration was determined by the method of Smith er ol. (1985), using the bicinchoninic acid protein assay reagent from Pierce Chemical Co. (Rockford, Ill.). Protein concentration of neutrophil suspension was 860 * 23 (mean f SD) pg protein/IO’ cell eq. RESULTS
Table 1 shows effects of ampicillin on the reduction of cytochrome c and on the superoxide generation of human neutrophils stimulated by PMA. Intact cells produced a superoxide dismutase-inhibitable reduction of cytochrome c by 0.09 + 0.02 (mean + SD) nmol/min/2 x IO5cell eq and a superoxide dismutasenoninhibitable reduction of cytochrome c by 0.06 f 0.02 (mean f SD) nmol/min/2 x IO’ cell eq.
1291
1292 Table
1.Effects of
ampicillin on cytochrome c reduction
cuvct1c
______-.
Refere”LX
Cytochrome c reduction (nmol/min)
Sampk
SOD SOD SOD SOD SOD SOD SOD SOD SOD SOD/PMA SOD/ABPC SOD/cells SOD/oclls/PMA SOD/alls/ABPC SOD/cells/PMA/ABPC
Mean f SD 0.03 * 0.01 2.08 + 0.03 0.06 f 0.02 0.03 f 0.01 2.72 f 0.04 0.16 f 0.03 2.83 f 0.04 2.92 f 0.05 5.84 f 0.04 0.03 * 0.01 0.70 2 0.02 0.09 f 0.02 2.88 f 0.03 0.75 f 0.04 3.76 + 0.05
SOD/PMA SOD/ABPC SOD/LXllS PMA ABPC Cells Cclls/PMA Cells/ABPC Cclls/PMA/ABPC PMA ABPC Cells Cells/PMA Cclls/ABPC Cells/PMA/ABPC
The assay method is described in Materials and Methods. All cuvct~cs contained HBSS buffer and cytochromc c. The results show” are mean f SD of thra different experiment. SOD, superoxide dismutasc.
PMA did not produce any significant changes in both superoxide dismutase-inhibitable and noninhibitable reduction of cytochrome c. However, neutrophils stimulated by 0.3 pg of PMA produced superoxide by 2.88 f 0.03 (mean f SD) nmol/min/2 x 10’ cell eq. On the other hand, 50 mM ampicillin amplified a superoxide dismutase-inhibitable reduction of cytochrome c by 0.70 + 0.02 (mean * SD) nmol/min/ 2 x 10’ cell eq and a superoxide dismutase-noninhibitable reduction of cytochrome c by 2.08 + 0.03 (mean f SD) nmol/min/2 x lo5 cell eq. However, ampicillin did not stimulate the superoxide production of resting cells and not amplify the superoxide production of neutrophils stimulated by PMA.
0
to
20
30
Concentration
40
50
of tmpici
60
I I in
TO
60
(mhl)
Fig. I. Dose-dependent effects of ampicillin on the reduction of cytochromc c. The assay method is described in Materials and Methods. The results shown are the mean f SD of three different
experiments.
Figure 1 shows dose-dependent effect of ampicillin on the reduction of cytochrome c which is superoxide dismutase-inhibitable and noninhibitable. Cytochrome c reduction increased in a linear fashion as an increasing concentration of ampicillin. DISCL’SSION
agents influence the function of phagocytic neutrophils (Hauser and Remington, 1982; Mandell, 1982). Briheim and Dahlgren (1987) demonstrated an inhibitory effect of penicillin G and ampicillin on luminol-enhanced chemiluminescence of neutrophils stimulated by a chemoattractant formylmethionyl-leucyl-phenylalanine. However, Duncker and Ullmann (1986) reported that ampicillin does not influence luminolenhanced chemiluminescence reaction of neutrophils stimulated by an opsonized zymosan. In the present study, the results obtained indicated no significant effects of ampicillin on the superoxide production of neutrophils stimulated by PMA (respiratory burst phenomenon). Ampicillin (Fig. 2) has been shown to be an antibiotic having microbicidal activity against both Gram-positive and negative bacteria (May and Delves, 1965). The results obtained here indicated that ampicillin may amplify both superoxide dismutase-inhibitable and noninhibitable reduction of cytochrome c, suggesting that this drug serves as an electron donor, like NAD(P)H and ascorbic acid, supplying superoxide dismutase or cytochrome c with an electron. The recent investigation by Scarpa et al. (1983) has demonstrated that one molecule of superoxide radical is generated for each molecule of ascorbic acid oxidized. Based on the evidence, it is considered that ampicillin, like ascorbic acid, generates the superoxide when it is oxidized. It is concluded that ampicillin may serve as an electron donor and/or a superoxide generator. It
is well known that antimicrobial
Acknowledgements-l J.T. Cumutte
Fig. 2. Structure of ampicillin (sodium a-aminobenzylpenicillin).
wish to thank Drs B.M. Babior and of the Research lnstitute of Scripps Clinic, La Jolla of the United States, for the encouragement given me lo undertake these studies and for their critical advice and review.
Ampicillin is an electron donor REFERENCES Babior B. M. (1978) Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298, 659-668. Briheim G. and Dahlgren C. (1987) Influence of antibiotics on formylmethionyl-leucyl-phenylalanine-induced leukocyte chemiluminescence. Antimicrob. Agents Chemother. 31, 763-767.
Duncker D. and Ullmann U. (1986) Influence of various antimicrobial agents on the chemiluminescence of phagocytosing human granulocytes. Chemotherapy 32, 18-24. Hauser W. E. Jr and Remington J. S. (1982) Effect of antibiotics on the immune response. Am. J. Med. 72, 71 I-716. Klebanoff S. J. (1975) Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hemarol. 12, 117-142.
Mandell L. A. (1982) Effect of antimicrobial and antineoplastic drugs on the phagocyte and microbicidal function of the polymorphonuclear leukocyte. Rm. infecr. Dis. 4, 683697. May J. R. and Delves D. M. (1965) Treatment of chronic bronchitis with ampicillin. Lancer 1, 929-933. McPhail L. C., Shirley P. S., Clayton C. C. and Snyderman R. (1985) Activation of the respiratory burst enzyme system from human neutrophils in a cell-free system. Evidence for a soluble cofactor. J. clin. Inuesr. 75, 1735-1739. Rossi F., Romeo D. and Patriarca P. (1972) Mechanism of phagocytosis-associated oxidative metabolism in poly-
1293
morphonuclear leucocytes and macrophages. J. rericuloendothel. Sot. 12, 127-149. Sbarra A. J. and Karnovsky M. L. (1959) The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J. biol. Chem. 234, 1355-I 362. Scarpa M.. Stevanato R., Viglino P. and Rigo A. (1983) Superoxide ion as active intermediate in the autoxidation of ascorbate by molecular oxygen: effect of superoxide dismutase. J. biol. Chem. 258, 6695497. Smith P. K., Krohn R. I., Hermanson G. T.. Mallia A. K.. Gartner F. H., Provenzano M. D., Fujimoto E. K.. Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyr. Eiochem.
150. 76-85.
Stossel T. P. (1974) Phagccytosis. N. Engl. J. Med. 290, 7 I7--723, 774 -780, 833-839. Umeki S. (1990) Human neutrophil cytosolic activation factor of the NADPH oxidase: characterization of activation kinetics. J. biol. Chem. 265, 5049-5054. Umeki S. and Soejima R. (1990) Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems. Biochim. biophys. Acra 1052, 21 I 215.
Yonetani T. (1965) Studies on cytochrome c peroxidase. II. Stoichiometry between enzyme, H,H,, and ferrocytochrome c and enzymic determination of extinction coefficients of cytochrome c. J. biol. Chem. 240, 4509 4514.