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Inhibition by amines indicates involvement of nitrogen dioxide in autocatalytic oxidation of oxyhemoglobin by nitrite
Biochimica et Biophysica Acta 871 (1986) 14-18 Elsevier
14
BBA 32520
Inhibition by amines indicates involvement of nitrogen dioxide in autocatalytic oxidation of oxyhemoglobin by nitrite Hiroaki Kosaka * and Mitsuro Uozumi Division of Environmental Health Research, Osaka Prefectural Institute of Public Health, 69-3-1, Nakamichi, Higashinari-Ku, Osaka 537 (Japan)
(Received November 5th. 1985) (Revised manuscript received January 27th, 1986)
Key words: Methemoglobin; Nitrite; Nitrogen dioxide; Nitrosamine formation; Autocatalytic oxidation
Oxidation of oxyhemoglobin by nitrite is characterized by a lag period followed by an autocatalytic phase. The oxidation can be inhibited by the addition of morpholine, piperidine, triethanolamine or triethylamine (6 mM each). These amines are known to react with nitrogen dioxide to yield nitrosamine. Unexpectedly, aniline or aminopyrine (120/zM each) markedly inhibited the oxidation. These compounds, but not the other amines given above, inhibited the peroxide compound formation from methemoglobin and hydrogen peroxide. The results establish that, during the oxidation, the peroxide compound is generated and converts nitrite into nitrogen dioxide by its peroxidatic activity, resulting in an autocatalytic phase.
Introduction Addition of nitrite to a solution of oxyhemoglobin transforms the hemoglobin into methemoglobin. The kinetics are characterized by a lag phase followed by autocatalysis [1 6]. However, the detailed mechanism of the reaction has not been determined. During the oxidation, we detected by the ESR technique a free radical similar to the methemoglobin free radical which is generated from methemoglobin and hydrogen peroxide [7]. The methemoglobin free radical is considered to be ferrylhemoglobin containing a free radical [8]. This oxidation was inhibited by addition of catalase but not by addition of superoxide dismutase [7]. Thus we speculated that the electron from nitrite and an electron from herne iron must both be taken up by the bound dioxygen to yield hydrogen peroxide and methemoglobin [7]. Hydrogen peroxide and methemoglobin react * To whom correspondence should be addressed.
to form a spectrophotometrically detectable peroxide compound (ferrylhemoglobin) [9]. The peroxide compound must catalyze the conversion of nitrite to nitrogen dioxide [10,11]. Although nitrogen dioxide must oxidize oxyhemoglobin, whether nitrogen dioxide is produced during the oxidation remains uncertain. Nitrogen dioxide (NO2) or its dimer (N204), but not nitric oxide, reacts with amines to yield diazo and N-nitrosamine products even under aqueous neutral and alkaline conditions [12-15]. Challis [13] reported rapid nitrosation of amino compounds by gaseous N 2 0 3 and N204 under aqueous neutral and alkaline conditions as follows. Nitrosation by gaseous N 2 0 3 and N 2 0 4 in aqueous solution is a recent finding probably because both had been expected to undergo rapid hydrolysis at pH > 5 to innocuous nitrite. Hydrolysis does occur, but less rapidly than the nitrosation of many amines. N203 and N 2 0 4 r e a c t about 2000times more rapidly with most amines than with H20. Iqbal et al. [12] detected nitrosomorpholine
0167-4838/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)
15
in mice that had received morpholine and inhaled nitrogen dioxide. In a previous study [15], we detected 15 n g / g nitrosodimethylamine from the blood of a rabbit that had received aminopyrine by intravenous dripping and inhaled 50 /~1/1 nitrogen dioxide gas. To investigate the species which oxidizes oxyhemoglobin, we added morpholine, piperidine, triethanolamine and triethylamine to the reaction. If the species oxidizing oxyhemoglobin is nitrogen dioxide, the oxidation must be inhibited by amine addition.
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Human adult hemoglobin from fresh blood was freed from catalase by a CM-Sephadex column [17]. Methemoglobin was prepared by treating the oxyhemoglobin with 5 equiv, of ferricyanide at 37°C for 30 rain and the ferricyanide was removed with Dowex 1-8. The concentration of hemoglobin is given on a monomer basis. Spectrophotometric measurements were carried out with a Hitachi 556 recording spectrophotometer with cell compartments thermostatically controlled at 25°C. The formation of methemoglobin was followed by measuring the absorbance at 577 nm and that of the peroxide compound at 630 nm. Nitrite and nitrate concentrations after the oxidation were measured by a high-pressure liquid chromatography model Trirotar III (Japan Spectroscopic Co., Ltd.) with a 25-cm-long, 4.6-mm-i.d. Du Pont Zipax SAX (strong anion exchange) column, because colorimetric determination of nitrite [18] was impossible in the presence of 6 mM morpholine. Separation of the sample components was monitored with a UVIDEC 100-III variablewavelength column monitor at 210 nm, the output terminals of which were connected to a recorder. The carrier buffer (flow rate 4 ml/min) was made from 2.44 mM N a H C O 3 and 0.56 mM K2CO 3. To avoid artifact formation of nitrosomorpholine, the sample solution (10 /xl) was analyzed directly by high-pressure liquid chromatography without protein precipitation. Results
Addition of morpholine (6 mM) or piperidine (6 mM) inhibited the oxidation of oxyhemoglobin
0
5 10 Minutes Fig. 1. Amine inhibition of the oxidation of oxyhemoglobin (120 /tM) by nitrite (360 ttM) in 0.05 M phosphate buffer at pH 7.0. Curve (1) control; (2) 6 mM piperidine; (3) 6 mM morpholine; (4) 120/~M aniline; (5) 120 ~tM aminopyrine. by nitrite as shown in Fig. 1. Both amines are known to react with nitrogen dioxide to yield nitrosamine [13]. Triethanolamine (6 mM) or triethylamine (6 raM), which also reacts with nitrogen dioxide [14,13], inhibited the oxidation like morpholine or piperidine. Unexpectedly, aniline or
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Fig. 2. Inhibition by aniline or aminopyrine, but not by morpholine, piperidine, triethanolamine or triethylamine, of the peroxide compound formation from methemoglobin (120 /~M) and hydrogen peroxide (107 ~M) in 0.05 M phosphate buffer at pH 7.0. Curve (1) control; (2) 6 mM morpholine; (3) 6 mM piperidine; (4) 6 mM triethanolamine; (5) 6 mM triethylamine; (6) 120 /~M aniline; (7) 120 /tM aminopyrine; (8) 1.2 mM aminopyrine.
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Fig. 3. Similaritybetween the inhibition by 6 mM morpholine of the oxidation of oxyhemoglobin(120 /~M) by nitrite (360 /tM) and the oxidation with 270 /~M nitrite in 0.05 M phosphate buffer at pH 7.0. Curve (1) 360 #M nitrite; (2) 270 /~M nitrite; (3) 252 #M nitrite; (4) 234 #M nitrite; (5) 360 /~M nitrite and 6 mM morpholine. aminopyrine markedly inhibited the oxidation even at 120/~M. Aniline is reported by Challis [13] to react with nitrogen dioxide or its dimer. Aminopyrine is nitrosated by nitrogen dioxide [15]. The reaction of methemoglobin and hydrogen peroxide forms a spectrophotometrically detecta-
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Discussion
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ble red compound (peroxide compound, ferrylhemoglobin) [8,11]. We previously concluded that the peroxide compound is involved in the autocatalysis [7]. To discover why the oxidation was inhibited markedly only by aniline or aminopyrine (Fig. 1), we investigated the effects of these amines upon the peroxide compound. Its formation was markedly inhibited only by aniline (120 /~M) or aminopyrine (120 /~M, 1.2 mM), as shown in Fig. 2. Morpholine, piperidine, triethanolamine or triethylamine did not inhibit its generation even at 6 mM. Inhibition of the oxidation of oxyhemoglobin with nitrite by morpholine, piperidine, triethanolamine or triethylamine indicates that nitrogen dioxide is produced. However, nitrite also reacts with these amines to yield nitrosamine with the maximum reaction rate at pH 3.4 [16]. As shown in Fig. 3, the effect of a single addition of 270/~M nitrite to the oxidation was similar to the addition of 360 I~M nitrite plus 6 mM morpholine. If these amines also react with nitrite at pH 7 and if delay of the oxidation is due to the decrease in nitrite for the reaction with these amines, additional consumption of at least 90/~M nitritite is expected by addition of 6 mM morpholine because we had previously determined that nitrite is consumed and nitrate is produced, the respective amounts being equivalent to the ferriheme produced [18]. However, the amount of nitrite in the presence of morpholine (Fig. 4B) was not lower than that of nitrite in its absence (Fig. 4A).
z
Fig. 4. High-pressure liquid chromatograms of residual nitrite in the absence (A) and presence of 6 m M morpholine (B) after the oxidations. Chart speed was 1.0 c m / m i n . Experimental conditions were as given in Fig. 1.
The present study demonstrates that the species produced during the oxidation is nitrogen dioxide, which oxidizes oxyhemoglobin to methemoglobin. The oxidation could be inhibited by the addition of morpholine, piperidine, triethanolamine or triethylamine (6 mM each). These amines are known to react with nitrogen dioxide but not with nitric oxide [13]. Unexpectedly, addition of aniline or aminopyrine (120 #M each) remarkably inhibited the oxidation, although they are also known to react with nitrogen dioxide. The addition of these compounds, but not the other amines given above, inhibited the peroxide compound formation from
17
methemoglobin and hydrogen peroxide. Hemoglobin-catalyzed aniline hydroxylation [19] and oxidative demethylation of aminopyrine by the hemeprotein-hydrogen peroxide complex [20] have been reported, offering suggestions for our study on inhibition of the peroxide compound formation by aniline or aminopyrine. The deceleration by aniline or aminopyrine of oxyhemoglobin oxidation with nitrite (Fig. 1) must be due to inhibition of the peroxide compound generation (Fig. 2). Previously we reported [11] similar but weak inhibitions by Bistris salts of oxidation of oxyhemoglobin with nitrite and of the peroxide compound formation. The marked inhibitions by aniline or aminopyrine (Figs. 1 and 2) reinforce our previous conclusion [7] that the peroxide compound is involved in the autocatalysis. These results support the proposition [7] that the electron from nitrite and an electron from heme iron are both taken up by the bound dioxygen to yield hydrogen peroxide, methemoglobin and nitrogen dioxide (NO2): H b O 2 + N O 2 + 2 H + .-. Hb 3+ + N O 2 + H 2 0 2
The reaction of ferrimyoglobin (Mb3+) and hydrogen peroxide is known to produce a spectrophotometrically detectable red compound (peroxide compound, ferrylmyoglobin, Mb4+) [9] and a free radical intermediate (metmyoglobin free radical, Mb "4+) [21]. King and Winfield [8] suggested that Mb "4+, a compound containing quadrivalent iron and a free radical, is a precursor of Mb4+. The hydrogen peroxide and methemoglobin react to yield the methemoglobin free radical (Hb'4+) and the peroxide compound (Hb 4+) which then oxidize nitrite to nitrogen dioxide [7,11]. Hb 3+ + H202 -~ Hb'4+ + H 2 0 Hb "4+ + N O 2 + H + --~ Hb 4+ + N O 2 Hb 4+ + N O 2 + H + ~ Hb 3+ + N O 2 + H 2 0
Nitrogen dioxide or its dimer oxidizes oxyhemoglobin to methemoglobin [22], forming an autocatalytic phase. The inhibition of the oxidation by morpholine or the other amines suggests the involvement of nitrogen dioxide. However, we must consider the
possibility that the decrease of nitrite due to the reaction with the amines delayed the oxidation, since these amines also react with nitrite. Mirvish [16] reported that morpholine leads to the maximum rate in the reaction with nitrite at pH 3.4, where the formation rate of nitrosomorpholine is 0.42 M 2. s-a. We calculated that 6 mM morpholine and 360 t~M nitrite yield nitrosomorpholine at a rate of 0.02 ~ M / m i n at pH 3.4. The amount of nitrite consumed in the reaction with morpholine at pH 7 must be much less than that at pH 3.4, because the nitrosating agents from nitrite are generated only under acidic conditions [13]. In addition, inhibition of the oxidation of oxyhemoglobin by 360/~M nitrite with the addition of 6 mM morpholine was simulated by a single addition of 270/~M nitrite (Fig. 3). Thus, additional consumption of at least 90 ~M nitrite must be detected after the oxidation by the addition of morpholine, if the delay of the oxidation by the addition of morpholine is due to the decrease of nitrite. But such a decrease of nitrite was not detected after addition of morpholine (Fig. 4). Therefore, the reason for the delayed oxidation after addition of morpholine is not that the nitrite for the reaction with morpholine decreased but because morpholine competed with oxyhemoglobin for the reaction with nitrogen dioxide. The present study on the oxidation of oxyhemoglobin by nitrite indicates that this reaction produces nitrogen dioxide, which oxidizes oxyhemoglobin to methemoglobin. Our present findings support our previous conclusion that this reaction generates the peroxide compound which converts nitrite into nitrogen dioxide, thus accelerating the oxidation.
Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References 1 Meier, R. (1925) Arch. Exp. Path. Pharmakol. 110, 241-264 2 Jung, F. and Remmer, H. (1949) Arch. Exp. Path. Pharmakol. 206, 459-474 3 Tomoda, A., Tsuji, A. and Yoneyama, Y. (1981) Biochem. J. 193, 169-179
18 4 Jung, F., Lassmann, G. and Ebert, B. (1981) Stud. Biophys. 85, 139-140 5 Ebert, B., Jung, F. and Lassmann, G. (1982) Stud. Biophys. 89, 65-69 6 Ebert, B., Lassmann, G. and Jung, F. (1983) Biomed. Biochem. Acta 42, 11/12, 154-158 7 Kosaka, H., Imaizumi, K. and Tyuma, I. (1982) Biochim. Biophys. Acta 702, 237-241 8 King, N.K. and Winfield, M.E. (1963) J. Biol. Chem. 238, 1520-1528 9 Keilin, D. and Hartree, E.F. (1935) Proc. R. Soc. Lond. Ser. B, 117, 1-15 10 Keiline, D. and Hartree, E.F. (1955) Biochem. J. 60, 310-325 11 Kosaka, H. and Tyuma, I. (1982) Biochim. Biophys. Acta 709, 187-193 12 lqbal, Z.M., Dahl, K. and Epstein, S.S. (1980) Science 207, 1475-1477
13 Challis, B.C. (1981) in Safety Evaluation of Nitrosatable drugs and Chemicals (Gibson, G.G. and loannides, C., eds.), pp. 16-55, Taylor & Francis Ltd., London 14 Gold, A. (1977) Anal. Chem. 49, 1448-1450 15 Kosaka, H., Uozumi, M. and Nakajima, T. (1984) Int. Arch. Occup. Environ. Health 54, 233-239 16 Mirvish, S.S. (1975) Toxicol. Appl. Pharmacol. 31,325-351 17 Winterhalter, K.H. and Huehns, E.R. (1964) J. Biol. Chem. 239, 3699-3705 18 Kosaka, H., lmaizumi, K., Imai, K. and Tyuma, I. (1979) Biochim. Biophys. Acta 581, 184-188 19 Mieyal, J.J. and Blumer, J.L. (1976) J. Biol. Chem. 251, 3442-3446 20 Griffin, B.W. (1978) Arch. Biochem. Biophys. 190, 850-853 21 Gibson, J.F. and Ingrain, D.J.E. (1956) Nature 178, 871-872 22 Chiodi, H., Collier, C.R. and Mohler, J.G. (1983) Environ. Res. 30, 9-15
14
BBA 32520
Inhibition by amines indicates involvement of nitrogen dioxide in autocatalytic oxidation of oxyhemoglobin by nitrite Hiroaki Kosaka * and Mitsuro Uozumi Division of Environmental Health Research, Osaka Prefectural Institute of Public Health, 69-3-1, Nakamichi, Higashinari-Ku, Osaka 537 (Japan)
(Received November 5th. 1985) (Revised manuscript received January 27th, 1986)
Key words: Methemoglobin; Nitrite; Nitrogen dioxide; Nitrosamine formation; Autocatalytic oxidation
Oxidation of oxyhemoglobin by nitrite is characterized by a lag period followed by an autocatalytic phase. The oxidation can be inhibited by the addition of morpholine, piperidine, triethanolamine or triethylamine (6 mM each). These amines are known to react with nitrogen dioxide to yield nitrosamine. Unexpectedly, aniline or aminopyrine (120/zM each) markedly inhibited the oxidation. These compounds, but not the other amines given above, inhibited the peroxide compound formation from methemoglobin and hydrogen peroxide. The results establish that, during the oxidation, the peroxide compound is generated and converts nitrite into nitrogen dioxide by its peroxidatic activity, resulting in an autocatalytic phase.
Introduction Addition of nitrite to a solution of oxyhemoglobin transforms the hemoglobin into methemoglobin. The kinetics are characterized by a lag phase followed by autocatalysis [1 6]. However, the detailed mechanism of the reaction has not been determined. During the oxidation, we detected by the ESR technique a free radical similar to the methemoglobin free radical which is generated from methemoglobin and hydrogen peroxide [7]. The methemoglobin free radical is considered to be ferrylhemoglobin containing a free radical [8]. This oxidation was inhibited by addition of catalase but not by addition of superoxide dismutase [7]. Thus we speculated that the electron from nitrite and an electron from herne iron must both be taken up by the bound dioxygen to yield hydrogen peroxide and methemoglobin [7]. Hydrogen peroxide and methemoglobin react * To whom correspondence should be addressed.
to form a spectrophotometrically detectable peroxide compound (ferrylhemoglobin) [9]. The peroxide compound must catalyze the conversion of nitrite to nitrogen dioxide [10,11]. Although nitrogen dioxide must oxidize oxyhemoglobin, whether nitrogen dioxide is produced during the oxidation remains uncertain. Nitrogen dioxide (NO2) or its dimer (N204), but not nitric oxide, reacts with amines to yield diazo and N-nitrosamine products even under aqueous neutral and alkaline conditions [12-15]. Challis [13] reported rapid nitrosation of amino compounds by gaseous N 2 0 3 and N204 under aqueous neutral and alkaline conditions as follows. Nitrosation by gaseous N 2 0 3 and N 2 0 4 in aqueous solution is a recent finding probably because both had been expected to undergo rapid hydrolysis at pH > 5 to innocuous nitrite. Hydrolysis does occur, but less rapidly than the nitrosation of many amines. N203 and N 2 0 4 r e a c t about 2000times more rapidly with most amines than with H20. Iqbal et al. [12] detected nitrosomorpholine
0167-4838/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)
15
in mice that had received morpholine and inhaled nitrogen dioxide. In a previous study [15], we detected 15 n g / g nitrosodimethylamine from the blood of a rabbit that had received aminopyrine by intravenous dripping and inhaled 50 /~1/1 nitrogen dioxide gas. To investigate the species which oxidizes oxyhemoglobin, we added morpholine, piperidine, triethanolamine and triethylamine to the reaction. If the species oxidizing oxyhemoglobin is nitrogen dioxide, the oxidation must be inhibited by amine addition.
4
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Human adult hemoglobin from fresh blood was freed from catalase by a CM-Sephadex column [17]. Methemoglobin was prepared by treating the oxyhemoglobin with 5 equiv, of ferricyanide at 37°C for 30 rain and the ferricyanide was removed with Dowex 1-8. The concentration of hemoglobin is given on a monomer basis. Spectrophotometric measurements were carried out with a Hitachi 556 recording spectrophotometer with cell compartments thermostatically controlled at 25°C. The formation of methemoglobin was followed by measuring the absorbance at 577 nm and that of the peroxide compound at 630 nm. Nitrite and nitrate concentrations after the oxidation were measured by a high-pressure liquid chromatography model Trirotar III (Japan Spectroscopic Co., Ltd.) with a 25-cm-long, 4.6-mm-i.d. Du Pont Zipax SAX (strong anion exchange) column, because colorimetric determination of nitrite [18] was impossible in the presence of 6 mM morpholine. Separation of the sample components was monitored with a UVIDEC 100-III variablewavelength column monitor at 210 nm, the output terminals of which were connected to a recorder. The carrier buffer (flow rate 4 ml/min) was made from 2.44 mM N a H C O 3 and 0.56 mM K2CO 3. To avoid artifact formation of nitrosomorpholine, the sample solution (10 /xl) was analyzed directly by high-pressure liquid chromatography without protein precipitation. Results
Addition of morpholine (6 mM) or piperidine (6 mM) inhibited the oxidation of oxyhemoglobin
0
5 10 Minutes Fig. 1. Amine inhibition of the oxidation of oxyhemoglobin (120 /tM) by nitrite (360 ttM) in 0.05 M phosphate buffer at pH 7.0. Curve (1) control; (2) 6 mM piperidine; (3) 6 mM morpholine; (4) 120/~M aniline; (5) 120 ~tM aminopyrine. by nitrite as shown in Fig. 1. Both amines are known to react with nitrogen dioxide to yield nitrosamine [13]. Triethanolamine (6 mM) or triethylamine (6 raM), which also reacts with nitrogen dioxide [14,13], inhibited the oxidation like morpholine or piperidine. Unexpectedly, aniline or
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Fig. 2. Inhibition by aniline or aminopyrine, but not by morpholine, piperidine, triethanolamine or triethylamine, of the peroxide compound formation from methemoglobin (120 /~M) and hydrogen peroxide (107 ~M) in 0.05 M phosphate buffer at pH 7.0. Curve (1) control; (2) 6 mM morpholine; (3) 6 mM piperidine; (4) 6 mM triethanolamine; (5) 6 mM triethylamine; (6) 120 /~M aniline; (7) 120 /tM aminopyrine; (8) 1.2 mM aminopyrine.
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Fig. 3. Similaritybetween the inhibition by 6 mM morpholine of the oxidation of oxyhemoglobin(120 /~M) by nitrite (360 /tM) and the oxidation with 270 /~M nitrite in 0.05 M phosphate buffer at pH 7.0. Curve (1) 360 #M nitrite; (2) 270 /~M nitrite; (3) 252 #M nitrite; (4) 234 #M nitrite; (5) 360 /~M nitrite and 6 mM morpholine. aminopyrine markedly inhibited the oxidation even at 120/~M. Aniline is reported by Challis [13] to react with nitrogen dioxide or its dimer. Aminopyrine is nitrosated by nitrogen dioxide [15]. The reaction of methemoglobin and hydrogen peroxide forms a spectrophotometrically detecta-
0 -i q~
/l il
A
Discussion
;D UI "0 0 J U1
7 ¥
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3 z
ble red compound (peroxide compound, ferrylhemoglobin) [8,11]. We previously concluded that the peroxide compound is involved in the autocatalysis [7]. To discover why the oxidation was inhibited markedly only by aniline or aminopyrine (Fig. 1), we investigated the effects of these amines upon the peroxide compound. Its formation was markedly inhibited only by aniline (120 /~M) or aminopyrine (120 /~M, 1.2 mM), as shown in Fig. 2. Morpholine, piperidine, triethanolamine or triethylamine did not inhibit its generation even at 6 mM. Inhibition of the oxidation of oxyhemoglobin with nitrite by morpholine, piperidine, triethanolamine or triethylamine indicates that nitrogen dioxide is produced. However, nitrite also reacts with these amines to yield nitrosamine with the maximum reaction rate at pH 3.4 [16]. As shown in Fig. 3, the effect of a single addition of 270/~M nitrite to the oxidation was similar to the addition of 360 I~M nitrite plus 6 mM morpholine. If these amines also react with nitrite at pH 7 and if delay of the oxidation is due to the decrease in nitrite for the reaction with these amines, additional consumption of at least 90/~M nitritite is expected by addition of 6 mM morpholine because we had previously determined that nitrite is consumed and nitrate is produced, the respective amounts being equivalent to the ferriheme produced [18]. However, the amount of nitrite in the presence of morpholine (Fig. 4B) was not lower than that of nitrite in its absence (Fig. 4A).
z
Fig. 4. High-pressure liquid chromatograms of residual nitrite in the absence (A) and presence of 6 m M morpholine (B) after the oxidations. Chart speed was 1.0 c m / m i n . Experimental conditions were as given in Fig. 1.
The present study demonstrates that the species produced during the oxidation is nitrogen dioxide, which oxidizes oxyhemoglobin to methemoglobin. The oxidation could be inhibited by the addition of morpholine, piperidine, triethanolamine or triethylamine (6 mM each). These amines are known to react with nitrogen dioxide but not with nitric oxide [13]. Unexpectedly, addition of aniline or aminopyrine (120 #M each) remarkably inhibited the oxidation, although they are also known to react with nitrogen dioxide. The addition of these compounds, but not the other amines given above, inhibited the peroxide compound formation from
17
methemoglobin and hydrogen peroxide. Hemoglobin-catalyzed aniline hydroxylation [19] and oxidative demethylation of aminopyrine by the hemeprotein-hydrogen peroxide complex [20] have been reported, offering suggestions for our study on inhibition of the peroxide compound formation by aniline or aminopyrine. The deceleration by aniline or aminopyrine of oxyhemoglobin oxidation with nitrite (Fig. 1) must be due to inhibition of the peroxide compound generation (Fig. 2). Previously we reported [11] similar but weak inhibitions by Bistris salts of oxidation of oxyhemoglobin with nitrite and of the peroxide compound formation. The marked inhibitions by aniline or aminopyrine (Figs. 1 and 2) reinforce our previous conclusion [7] that the peroxide compound is involved in the autocatalysis. These results support the proposition [7] that the electron from nitrite and an electron from heme iron are both taken up by the bound dioxygen to yield hydrogen peroxide, methemoglobin and nitrogen dioxide (NO2): H b O 2 + N O 2 + 2 H + .-. Hb 3+ + N O 2 + H 2 0 2
The reaction of ferrimyoglobin (Mb3+) and hydrogen peroxide is known to produce a spectrophotometrically detectable red compound (peroxide compound, ferrylmyoglobin, Mb4+) [9] and a free radical intermediate (metmyoglobin free radical, Mb "4+) [21]. King and Winfield [8] suggested that Mb "4+, a compound containing quadrivalent iron and a free radical, is a precursor of Mb4+. The hydrogen peroxide and methemoglobin react to yield the methemoglobin free radical (Hb'4+) and the peroxide compound (Hb 4+) which then oxidize nitrite to nitrogen dioxide [7,11]. Hb 3+ + H202 -~ Hb'4+ + H 2 0 Hb "4+ + N O 2 + H + --~ Hb 4+ + N O 2 Hb 4+ + N O 2 + H + ~ Hb 3+ + N O 2 + H 2 0
Nitrogen dioxide or its dimer oxidizes oxyhemoglobin to methemoglobin [22], forming an autocatalytic phase. The inhibition of the oxidation by morpholine or the other amines suggests the involvement of nitrogen dioxide. However, we must consider the
possibility that the decrease of nitrite due to the reaction with the amines delayed the oxidation, since these amines also react with nitrite. Mirvish [16] reported that morpholine leads to the maximum rate in the reaction with nitrite at pH 3.4, where the formation rate of nitrosomorpholine is 0.42 M 2. s-a. We calculated that 6 mM morpholine and 360 t~M nitrite yield nitrosomorpholine at a rate of 0.02 ~ M / m i n at pH 3.4. The amount of nitrite consumed in the reaction with morpholine at pH 7 must be much less than that at pH 3.4, because the nitrosating agents from nitrite are generated only under acidic conditions [13]. In addition, inhibition of the oxidation of oxyhemoglobin by 360/~M nitrite with the addition of 6 mM morpholine was simulated by a single addition of 270/~M nitrite (Fig. 3). Thus, additional consumption of at least 90 ~M nitrite must be detected after the oxidation by the addition of morpholine, if the delay of the oxidation by the addition of morpholine is due to the decrease of nitrite. But such a decrease of nitrite was not detected after addition of morpholine (Fig. 4). Therefore, the reason for the delayed oxidation after addition of morpholine is not that the nitrite for the reaction with morpholine decreased but because morpholine competed with oxyhemoglobin for the reaction with nitrogen dioxide. The present study on the oxidation of oxyhemoglobin by nitrite indicates that this reaction produces nitrogen dioxide, which oxidizes oxyhemoglobin to methemoglobin. Our present findings support our previous conclusion that this reaction generates the peroxide compound which converts nitrite into nitrogen dioxide, thus accelerating the oxidation.
Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References 1 Meier, R. (1925) Arch. Exp. Path. Pharmakol. 110, 241-264 2 Jung, F. and Remmer, H. (1949) Arch. Exp. Path. Pharmakol. 206, 459-474 3 Tomoda, A., Tsuji, A. and Yoneyama, Y. (1981) Biochem. J. 193, 169-179
18 4 Jung, F., Lassmann, G. and Ebert, B. (1981) Stud. Biophys. 85, 139-140 5 Ebert, B., Jung, F. and Lassmann, G. (1982) Stud. Biophys. 89, 65-69 6 Ebert, B., Lassmann, G. and Jung, F. (1983) Biomed. Biochem. Acta 42, 11/12, 154-158 7 Kosaka, H., Imaizumi, K. and Tyuma, I. (1982) Biochim. Biophys. Acta 702, 237-241 8 King, N.K. and Winfield, M.E. (1963) J. Biol. Chem. 238, 1520-1528 9 Keilin, D. and Hartree, E.F. (1935) Proc. R. Soc. Lond. Ser. B, 117, 1-15 10 Keiline, D. and Hartree, E.F. (1955) Biochem. J. 60, 310-325 11 Kosaka, H. and Tyuma, I. (1982) Biochim. Biophys. Acta 709, 187-193 12 lqbal, Z.M., Dahl, K. and Epstein, S.S. (1980) Science 207, 1475-1477
13 Challis, B.C. (1981) in Safety Evaluation of Nitrosatable drugs and Chemicals (Gibson, G.G. and loannides, C., eds.), pp. 16-55, Taylor & Francis Ltd., London 14 Gold, A. (1977) Anal. Chem. 49, 1448-1450 15 Kosaka, H., Uozumi, M. and Nakajima, T. (1984) Int. Arch. Occup. Environ. Health 54, 233-239 16 Mirvish, S.S. (1975) Toxicol. Appl. Pharmacol. 31,325-351 17 Winterhalter, K.H. and Huehns, E.R. (1964) J. Biol. Chem. 239, 3699-3705 18 Kosaka, H., lmaizumi, K., Imai, K. and Tyuma, I. (1979) Biochim. Biophys. Acta 581, 184-188 19 Mieyal, J.J. and Blumer, J.L. (1976) J. Biol. Chem. 251, 3442-3446 20 Griffin, B.W. (1978) Arch. Biochem. Biophys. 190, 850-853 21 Gibson, J.F. and Ingrain, D.J.E. (1956) Nature 178, 871-872 22 Chiodi, H., Collier, C.R. and Mohler, J.G. (1983) Environ. Res. 30, 9-15