- Email: [email protected]
H2O2 production during NADPH oxidation by the granule fraction of phagocytosing polymorphonuclear leucocytes
SHORT COMMUNICATIONS BBA
20I
23512
H202 production during NADPH oxidation by the granule fraction of phagocytosing polymorphonuclear leucocytes I t is known that H20 ~is formed in phagocytosing polymorphonuclear leucocytes during the period of accelerated metabolism 1-~. Recently we have obtained evidences that an appreciable amount of the peroxide accumulate only when 2 mM KCN is present 4. Previous work in our laboratory has shown that the stimulation of respiration and the modification of carbohydrate metabolism of polymorphonuclear leucocytes induced b y phagocytosis or by treatment with surface-active agents are linked to a direct oxidation of N A D P H by increased activity of intragranular N A D P H oxidase 5-14, In the present study the relationships between the formation, accumulation and utilization of H20 2 and the activities of the enzymes of the granule fraction of phagocytosing polymorphonuclear leucocytes have been investigated. The granules were obtained from phagocytosing gninea-pig polymorphonuclear leucocytes and the oxidation of N A D P H was measured as oxygen uptake with a Clark oxygen electrode at 37 ° as previously described 5,11,14. The H~O 2 was measured as oxygen evolved ~, 15 after the addition of catalase (Sigma).
NADM -- ~
KCN
NA~°HI
A
,..
r
CATALASE
(pro1.37
~.
B
60 sec
Fig. I. O x y g e n traces of the oxidation of N A D P H and of the decomposition of H202 a c c u m u l a t e d b y the granule fraction of p o l y m o r p h o n u c l e a r leucocytes (2 mg of protein). The s y s t e m was as follows: 14o mM sucrose, 36 mM p h o s p h a t e buffer (pH 7.2) ; 19 mM KC1; 1. 5 mM MgCI~; o.8 mM N A D P H ; 2 mM KCN (Trace A) ; catalase 6.5" lO3 Sigma U. Total volume 1.8 ml. T e m p e r a t u r e 37 °. The points a and b (dotted arrows) indicate the drawing of 5 ° / z l for the s p e c t r o p h o t o m e t r i c m e a s u r e m e n t of N A D P H and its total a m o u n t s in the s y s t e m are given in brackets. Trace A: oxygen u p t a k e between a and b o.21 F a t o m ; N A D P H oxidized between a and b o . i o #mole; o x y g e n liberated after catalase o . i i u a t o m . Trace B: oxygen u p t a k e b e t w e e n a and b o.13 # a t o m ; N A D P H oxidized between a and b o.14 umole.
The experiments presented in Fig. I and in Table I show that catalase causes a rapid release of oxygen only when the granules of phagocytosing polymorphonuclear leucocytes are oxidizing N A D P H in the presence of 2 mM KCN. The ratio vmoles of oxygen consumed//~moles of H~O~ accumulated approximates to unity, suggesting that almost all the oxygen is consumed with accumulation of H20 2. Moreover, it is worthy to point out that the rate of oxygen uptake before the addition of catalase was significantly lower in absence of KCN, as one might expect if H~O 2 is decomposed Biochim. Biophys. Acta, 184 (1969) 2 o i - 2 o 3
202
SHORT COMMUNICATIONS
TABLE I O X Y G E N U P T A K E , N A D P H O X I D I Z E D AND H 2 0 ~ PRODUCED BY THE GRANULE FRACTION (2 ITlg PROTEIN) OF PHAGOCYTOSING LEUCOCYTES
For conditions see Fig. 1 and t e x t (average values from 14 experiments).
Oxygen u p t a k e (/zatoms) N A D P H oxidized (/,moles) H202 produced (/*moles) R a t i o / * a t o m s O[/~moles N A D P H * Ratio/~moles O2]/~moles H202 R a t i o / , m o l e s N A D P H ] / , m o l e s H202"*
Without K C N
With K C N
o.124 o.162 -0.76 ---
2.18 o.142 o.o91 1.53 1.2o 1.56
* Theoretical ratio ~ 1.0 w h e n HzO 2 is not accumulated and 2.0 when H202 is accumulated. ** Theoretical ratio ~ 1.0 w h e n N A D P H is oxidized with formation of H~Oe and the latter is all accumulated.
by endogenous catalase and if the latter is inhibited by 2 mM KCN. When I mM KCN was used endogenous catalase was not inhibited 1~. The fact that H,O 2 is produced during the oxidation of NADPH by the granule fraction of phagocytosing polymorphonuclear leucocytes and that the peroxide accumulates when 2 mM KCN is present, raises the problem of the stoichiometric relations between the amount of NADPH oxidized, the oxygen consumed and the H202 accumulated. The amount of NADPH oxidized was measured by spectrophotometric determination on samples (50 vl) drawn at various intervals during the recording of the oxygen uptake. The results show that the ratio ~atoms of oxygen consumed per #moles of NADPH utilized approximates to unity in the absence of KCN (Fig. IB), while it is roughly doubled when H202 is accumulating (Fig. IA). The trace of the Fig. IA represents the maximal value we have Obtained among 14 experiments, while the mean values of all the experiments are given in Table I. It can be seen that the ratio oxygen/NADPH was higher in the presence of 2 mM KCN. However, in both conditions (i.e. with and without KCN) this ratio was frequently lower than the theoretical one. The results (Table I) also show an experimental ratio/,moles of NADPH oxidized/~moles of H~O 2 accumulated higher than the theoretical one. These data may be interpreted as indicating that an aliquot of NADPH (approx. 20 %) is oxidized through reaction(s) independent of oxygen. The results presented here also allow to discuss the mechanisms responsible for the utilization of H20 ~ formed in the absence of KCN. Two mechanisms could be postulated: (I) a peroxidatic onO 6-1s, whereby H20, is utilized for the oxidation of a reduced compound. Since the ratio ~atoms oxygen/tzmoles NADPH in absence of KCN is not higher than 1.0, one must assume that the reduced compound oxidized by the peroxidase could be NADPH. (2) A catalatic mechanism, whereby the H~O2 formed during the oxidation of NADPH is converted into H20 and i/2 02. In both cases the final stoichiometry is I ~atom of oxygen per i ~mole of NADPH oxidized without accumulation of H202. The results presented in this communication clearly show that 2 mM KCN modifies the ratio ~atoms of oxygen//zmoles of NADPH oxidized. If the utilization of the peroxide occurs through a peroxidatic reaction, the KCN should modify the ratio by decreasing the amount of NADPH oxidized without Biochim. Biophys. Acta, 184 (1969) 2Ol-2O3
SHORT COMMUNICATIONS
203
modification of oxygen uptake. On the contrary if a catalatic reaction is operating, the KCN should change the ratio by increasing the oxygen uptake without modification of the amount of NADPH oxidized. Our results show that 2 mM KCN mainly modifies the oxygen uptake indicating that the H202 formed during the oxidation of NADPH by the granule fraction of phagocytosing polymorphonuclear leucocytes is preferentially utilized through a catalatic reaction. The mechanism, which is actually operating in phagocytosing cells, remains to be ascertained. Besides the significance in protecting the cells from any toxic effects of H202 (ref. I9), myeloperoxidase has been suggested to be an important factor for the bactericidal activity of polymorphonuclear leucocytes ~-26. The fact that in the conditions employed here in vitro a catalatic mechanism seems to be mainly involved in H20 2 destruction, while the hypothesis of a bactericidal effect requires the activity of peroxidatic reactions, does not exclude that the two mechanisms operate in vivo in different and mutable proportions in relation to an endogenous control depending on the (experimental) conditions of phagocytosis as well as on the functional exigency of the phagocyte and on the amount of the peroxide formed.
Institute of General Pathology, University of Trieste (Italy), and Institute of General Pathology, University of Padova (Italy) I 2 3 4 5 6 7 8 9 to 11 I2 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F. Rossi M. ZATTI P. PATRIARCA
G. Y. N. IYBR, M. F. ISLAM AND J. H. QUASTEL, Nature, 192 (1961) 535. J. ROBERTS AND Z. CAMACHO, Nature, 216 (1967) 606. B. PAUL AND A. J. SBARRA, Biochim. Biophys. Mcta, 156 (1968) 168. M. ZATTI, F. R o s s I AND P. PATRIARCA, Experientia, 24 (1968) 669. F. R o s s I ANO M. ZATTI, Brit. J. Exptl. Pathol., 45 (1964) 548. F. R o s s I AND M. ZATTI, Experientia, 20 (1964) 21. M. ZATTI AND F. ROSSI, Biochim. Biophys. Acta, 99 (1965) 557. M. ZATTI, F. R o s s I AND V. ME,'~EGHELLI, J. Microscopie, 4 (1965) 95. M. ZATTI, F. ROSSI AND V. MENEGHELLI, Brit. J. Exptl. Pathol., 46 (1965) 227. F. ROSSl AND M. ZATTL Biochim. Biophys. Acta, 113 (1966) 395. F. R o s s I ANO M. ZATTI, Biochim. Biophys. Acta, 121 (1966) i i o . M. ZATTI AND F. ROSSI, Experientia, 22 (1966) 758. M. ZATTI AND F. ROSSI, Biochim. Biophys. Acta, 148 (1967) 553. F. R o s s I AND M. ZATTI, Biochim. Biophys. Aeta, 153 (1968) 296. M. I~ORTH AND P. K. JENSEN, Biochim. Biophys. Acta, 139 (1967) 171. J. ROBERTS AND J. I-I. QUASTEL, Nature, 202 (1964) 85. T. AKAZAWA AND E. E. CONN, J. Biol. Chem., 232 (1958) 402. J. B. MOOD AND R. ]ODRRIS, J. Biol. Chem., 234 (1959) 3281. M. RECHCIGL AND W. H. EVANS, Nature, 199 (1963) IOOI. S. J. KLEBANOFF AND R. G. LUEBKE, Proc. Soc. Exptl. Biol. Med., 118 (1965) 483 . I~. J. SELVARAJ AND A. J. SBARRA, Nature, 211 (1966) 1272. R. J. McRIPLE¥ AND A. J. SBARRA, J. Baeteriol., 94 (I967) 1417. R. J. McRIPLEY AND A. J. SBARRA, J. Bacteriol., 94 (1967) 1425. S. J. KLEBANOFF, W. H. CLEM AND R. G. LUEBKE, Biochim. Biophys. Acta, 117 (1966) 63. S. J. KLEBANOFF, J. Exptl. Med., 126 (1967) lO63. S. J. KLEBANOFF, J. Bacleriol., 95 (1968) 2131.
Received January 27th, 1969 Biochim. Biophys. Acta, 184 (1969) 2Ol-2O3
20I
23512
H202 production during NADPH oxidation by the granule fraction of phagocytosing polymorphonuclear leucocytes I t is known that H20 ~is formed in phagocytosing polymorphonuclear leucocytes during the period of accelerated metabolism 1-~. Recently we have obtained evidences that an appreciable amount of the peroxide accumulate only when 2 mM KCN is present 4. Previous work in our laboratory has shown that the stimulation of respiration and the modification of carbohydrate metabolism of polymorphonuclear leucocytes induced b y phagocytosis or by treatment with surface-active agents are linked to a direct oxidation of N A D P H by increased activity of intragranular N A D P H oxidase 5-14, In the present study the relationships between the formation, accumulation and utilization of H20 2 and the activities of the enzymes of the granule fraction of phagocytosing polymorphonuclear leucocytes have been investigated. The granules were obtained from phagocytosing gninea-pig polymorphonuclear leucocytes and the oxidation of N A D P H was measured as oxygen uptake with a Clark oxygen electrode at 37 ° as previously described 5,11,14. The H~O 2 was measured as oxygen evolved ~, 15 after the addition of catalase (Sigma).
NADM -- ~
KCN
NA~°HI
A
,..
r
CATALASE
(pro1.37
~.
B
60 sec
Fig. I. O x y g e n traces of the oxidation of N A D P H and of the decomposition of H202 a c c u m u l a t e d b y the granule fraction of p o l y m o r p h o n u c l e a r leucocytes (2 mg of protein). The s y s t e m was as follows: 14o mM sucrose, 36 mM p h o s p h a t e buffer (pH 7.2) ; 19 mM KC1; 1. 5 mM MgCI~; o.8 mM N A D P H ; 2 mM KCN (Trace A) ; catalase 6.5" lO3 Sigma U. Total volume 1.8 ml. T e m p e r a t u r e 37 °. The points a and b (dotted arrows) indicate the drawing of 5 ° / z l for the s p e c t r o p h o t o m e t r i c m e a s u r e m e n t of N A D P H and its total a m o u n t s in the s y s t e m are given in brackets. Trace A: oxygen u p t a k e between a and b o.21 F a t o m ; N A D P H oxidized between a and b o . i o #mole; o x y g e n liberated after catalase o . i i u a t o m . Trace B: oxygen u p t a k e b e t w e e n a and b o.13 # a t o m ; N A D P H oxidized between a and b o.14 umole.
The experiments presented in Fig. I and in Table I show that catalase causes a rapid release of oxygen only when the granules of phagocytosing polymorphonuclear leucocytes are oxidizing N A D P H in the presence of 2 mM KCN. The ratio vmoles of oxygen consumed//~moles of H~O~ accumulated approximates to unity, suggesting that almost all the oxygen is consumed with accumulation of H20 2. Moreover, it is worthy to point out that the rate of oxygen uptake before the addition of catalase was significantly lower in absence of KCN, as one might expect if H~O 2 is decomposed Biochim. Biophys. Acta, 184 (1969) 2 o i - 2 o 3
202
SHORT COMMUNICATIONS
TABLE I O X Y G E N U P T A K E , N A D P H O X I D I Z E D AND H 2 0 ~ PRODUCED BY THE GRANULE FRACTION (2 ITlg PROTEIN) OF PHAGOCYTOSING LEUCOCYTES
For conditions see Fig. 1 and t e x t (average values from 14 experiments).
Oxygen u p t a k e (/zatoms) N A D P H oxidized (/,moles) H202 produced (/*moles) R a t i o / * a t o m s O[/~moles N A D P H * Ratio/~moles O2]/~moles H202 R a t i o / , m o l e s N A D P H ] / , m o l e s H202"*
Without K C N
With K C N
o.124 o.162 -0.76 ---
2.18 o.142 o.o91 1.53 1.2o 1.56
* Theoretical ratio ~ 1.0 w h e n HzO 2 is not accumulated and 2.0 when H202 is accumulated. ** Theoretical ratio ~ 1.0 w h e n N A D P H is oxidized with formation of H~Oe and the latter is all accumulated.
by endogenous catalase and if the latter is inhibited by 2 mM KCN. When I mM KCN was used endogenous catalase was not inhibited 1~. The fact that H,O 2 is produced during the oxidation of NADPH by the granule fraction of phagocytosing polymorphonuclear leucocytes and that the peroxide accumulates when 2 mM KCN is present, raises the problem of the stoichiometric relations between the amount of NADPH oxidized, the oxygen consumed and the H202 accumulated. The amount of NADPH oxidized was measured by spectrophotometric determination on samples (50 vl) drawn at various intervals during the recording of the oxygen uptake. The results show that the ratio ~atoms of oxygen consumed per #moles of NADPH utilized approximates to unity in the absence of KCN (Fig. IB), while it is roughly doubled when H202 is accumulating (Fig. IA). The trace of the Fig. IA represents the maximal value we have Obtained among 14 experiments, while the mean values of all the experiments are given in Table I. It can be seen that the ratio oxygen/NADPH was higher in the presence of 2 mM KCN. However, in both conditions (i.e. with and without KCN) this ratio was frequently lower than the theoretical one. The results (Table I) also show an experimental ratio/,moles of NADPH oxidized/~moles of H~O 2 accumulated higher than the theoretical one. These data may be interpreted as indicating that an aliquot of NADPH (approx. 20 %) is oxidized through reaction(s) independent of oxygen. The results presented here also allow to discuss the mechanisms responsible for the utilization of H20 ~ formed in the absence of KCN. Two mechanisms could be postulated: (I) a peroxidatic onO 6-1s, whereby H20, is utilized for the oxidation of a reduced compound. Since the ratio ~atoms oxygen/tzmoles NADPH in absence of KCN is not higher than 1.0, one must assume that the reduced compound oxidized by the peroxidase could be NADPH. (2) A catalatic mechanism, whereby the H~O2 formed during the oxidation of NADPH is converted into H20 and i/2 02. In both cases the final stoichiometry is I ~atom of oxygen per i ~mole of NADPH oxidized without accumulation of H202. The results presented in this communication clearly show that 2 mM KCN modifies the ratio ~atoms of oxygen//zmoles of NADPH oxidized. If the utilization of the peroxide occurs through a peroxidatic reaction, the KCN should modify the ratio by decreasing the amount of NADPH oxidized without Biochim. Biophys. Acta, 184 (1969) 2Ol-2O3
SHORT COMMUNICATIONS
203
modification of oxygen uptake. On the contrary if a catalatic reaction is operating, the KCN should change the ratio by increasing the oxygen uptake without modification of the amount of NADPH oxidized. Our results show that 2 mM KCN mainly modifies the oxygen uptake indicating that the H202 formed during the oxidation of NADPH by the granule fraction of phagocytosing polymorphonuclear leucocytes is preferentially utilized through a catalatic reaction. The mechanism, which is actually operating in phagocytosing cells, remains to be ascertained. Besides the significance in protecting the cells from any toxic effects of H202 (ref. I9), myeloperoxidase has been suggested to be an important factor for the bactericidal activity of polymorphonuclear leucocytes ~-26. The fact that in the conditions employed here in vitro a catalatic mechanism seems to be mainly involved in H20 2 destruction, while the hypothesis of a bactericidal effect requires the activity of peroxidatic reactions, does not exclude that the two mechanisms operate in vivo in different and mutable proportions in relation to an endogenous control depending on the (experimental) conditions of phagocytosis as well as on the functional exigency of the phagocyte and on the amount of the peroxide formed.
Institute of General Pathology, University of Trieste (Italy), and Institute of General Pathology, University of Padova (Italy) I 2 3 4 5 6 7 8 9 to 11 I2 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F. Rossi M. ZATTI P. PATRIARCA
G. Y. N. IYBR, M. F. ISLAM AND J. H. QUASTEL, Nature, 192 (1961) 535. J. ROBERTS AND Z. CAMACHO, Nature, 216 (1967) 606. B. PAUL AND A. J. SBARRA, Biochim. Biophys. Mcta, 156 (1968) 168. M. ZATTI, F. R o s s I AND P. PATRIARCA, Experientia, 24 (1968) 669. F. R o s s I ANO M. ZATTI, Brit. J. Exptl. Pathol., 45 (1964) 548. F. R o s s I AND M. ZATTI, Experientia, 20 (1964) 21. M. ZATTI AND F. ROSSI, Biochim. Biophys. Acta, 99 (1965) 557. M. ZATTI, F. R o s s I AND V. ME,'~EGHELLI, J. Microscopie, 4 (1965) 95. M. ZATTI, F. ROSSI AND V. MENEGHELLI, Brit. J. Exptl. Pathol., 46 (1965) 227. F. ROSSl AND M. ZATTL Biochim. Biophys. Acta, 113 (1966) 395. F. R o s s I ANO M. ZATTI, Biochim. Biophys. Acta, 121 (1966) i i o . M. ZATTI AND F. ROSSI, Experientia, 22 (1966) 758. M. ZATTI AND F. ROSSI, Biochim. Biophys. Acta, 148 (1967) 553. F. R o s s I AND M. ZATTI, Biochim. Biophys. Aeta, 153 (1968) 296. M. I~ORTH AND P. K. JENSEN, Biochim. Biophys. Acta, 139 (1967) 171. J. ROBERTS AND J. I-I. QUASTEL, Nature, 202 (1964) 85. T. AKAZAWA AND E. E. CONN, J. Biol. Chem., 232 (1958) 402. J. B. MOOD AND R. ]ODRRIS, J. Biol. Chem., 234 (1959) 3281. M. RECHCIGL AND W. H. EVANS, Nature, 199 (1963) IOOI. S. J. KLEBANOFF AND R. G. LUEBKE, Proc. Soc. Exptl. Biol. Med., 118 (1965) 483 . I~. J. SELVARAJ AND A. J. SBARRA, Nature, 211 (1966) 1272. R. J. McRIPLE¥ AND A. J. SBARRA, J. Baeteriol., 94 (I967) 1417. R. J. McRIPLEY AND A. J. SBARRA, J. Bacteriol., 94 (1967) 1425. S. J. KLEBANOFF, W. H. CLEM AND R. G. LUEBKE, Biochim. Biophys. Acta, 117 (1966) 63. S. J. KLEBANOFF, J. Exptl. Med., 126 (1967) lO63. S. J. KLEBANOFF, J. Bacleriol., 95 (1968) 2131.
Received January 27th, 1969 Biochim. Biophys. Acta, 184 (1969) 2Ol-2O3