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Effect of NADH on the redox state of human hemoglobin
Clinica Chimica Acta 324 (2002) 129 – 134 www.elsevier.com/locate/clinchim
Effect of NADH on the redox state of human hemoglobin Robert A. Olek a, Jedrzej Antosiewicz a, Gian Carlo Caulini b, Giancarlo Falcioni b,* a
Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education Wiejska 1, 80-336 Gdansk, Poland b Department of Biology MCA, University of Camerino, Via Camerini 2, I-62032 Camerino, Italy Received 6 February 2002; received in revised form 20 June 2002; accepted 27 June 2002
Abstract In this work, we report that NADH can increase the autoxidation rate of hemoglobin (HbA) in a pH-dependent fashion. During this process, this cofactor is itself oxidized. The presence of superoxide dismutase (SOD) and/or catalase (CAT) can inhibit this result. At lower pH rates, the effect of NADH on the hemoglobin autoxidation rate is more enhanced; in addition, the rate of NADH oxidation is increased. Our data indicates that the reduced pyridine nucleotide may influence the redox state of human hemoglobin by a mechanism, which probably involves free radical species. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Hemoglobin; NADH; Catalase; Superoxide dismutase; ROS
1. Introduction The physiological function of hemoglobin is to transport oxygen to the tissues, and this depends on the ability of the ferrous form (Hb2 + ) to reversibly bind molecular oxygen. However, oxy-hemoglobin can turn to met-Hb (the Hb3 + form), which is unable to transport oxygen. This conversion is associated with superoxide anion production [1] and thereby with products such as hydrogen peroxide or hydroxyl radicals, which can derive from superoxide anion itself. The superoxide anion radical and hydrogen peroxide can further react with hemoglobin, contributing to the level of methemoglobin produced [2].
*
Corresponding author. Tel.: +39-737-403211; fax: +39-737636216. E-mail address: [email protected] (G. Falcioni).
About 3% of met-Hb is produced in the erythrocyte in 24 h [3], but its level is maintained constantly low (about 1%) by the presence of met-hemoglobin reductases—NADH and NADPH-dependent enzymes. It has been reported that reduced pyridine nucleotides are also capable of non-enzymatic reduction of metHb but require the presence of an electron carrier [4] or EDTA [5]. On the other hand, it is well established that NADH can be oxidized by reactive oxygen species [6].
:
NADH þ O2 þ Hþ ! NAD: þ H2 O2
:
NAD: þ O2 ! NAD þ O2
ð1Þ ð2Þ
In this study, we have investigated the influence of NADH on the redox state of HbA and the role of SOD and CAT in this process, which probably involves reactive oxygen species.
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 2 4 2 - 5
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R.A. Olek et al. / Clinica Chimica Acta 324 (2002) 129–134
Experiments were carried out in the presence of different chelating agents such as EDTA, diethylenetriaminopentaacetic acid (DTPA) and deferoxamine, which as metal chelates are involved in the formation and quenching of reactive oxygen species [7,8].
2. Materials and methods Human hemoglobin (HbA) was obtained as previously described [9]. Hemoglobin at a concentration of 1 mg/ml was incubated in 10 mmol/l Tris – HCl buffer, pH 7.4, or 50 mmol/l bis-Tris buffer pH 6.8 at 35 jC. Incubation mixtures contained EDTA at a final concentration of 0.1 mmol/l. Autoxidation was monitored in a Cary 219 spectrophotometer, equipped with a thermostatically controlled cell holder. Changes in the absorption spectrum ranging from 500 to 700 nm were recorded as a function of time. Reference value for complete oxidation was estimated by addition of potassium ferricyanide. NADH oxidation was monitored spectrophotometrically at 340 nm. All the compounds, NADH as well as the enzymes superoxide dismutase (SOD; EC 1.15.1.1)
and catalase (CAT; EC 1.11.1.6) were purchased from Sigma-Aldrich.
3. Results The effect of increasing the concentration of NADH on the kinetics of HbO2 autoxidation was investigated. The time course of hemoglobin oxidation during incubation at 35 jC and pH 7.4 is reported in Fig. 1. It appears that NADH has a concentration-dependent accelerating effect on autoxidation. It is well to point out that 0.1 mmol/l EDTA was present in the medium for chelating free metal ions. After 3 h of incubation, met-Hb reaches 5.5% in the control and almost 20% in the presence of 0.5 mmol/l NADH. The effect of NADH in accelerating met-hemoglobin formation rate was present also when we used other chelating agents such as deferoxamine and DTPA (data not shown). NAD + has no effect on the autoxidation rate (data not shown). In order to assess a possible involvement of reactive oxygen species in this process, the effect of superoxide dismutase and catalase was investigated. The results reported in Fig. 2 show that the presence of 0.1 Amol/l SOD in our experimental conditions does
Fig. 1. Effect of NADH on the time course of hemoglobin autoxidation: (*) control, (E) 0.1 mmol/l NADH, (n) 0.3 mmol/l NADH, (.) 0.5 mmol/l NADH. Hemoglobin (1 mg/ml) was incubated in 10 mmol/l Tris – HCl + 0.1 mmol/l EDTA pH 7.4 at 35 jC.
R.A. Olek et al. / Clinica Chimica Acta 324 (2002) 129–134
131
Fig. 2. Influence of superoxide dismutase (0.1 AM) on the rate of met-Hb formation in the presence and in the absence of NADH: (*) control, (E) 0.1 mmol/l NADH, (.) 0.1 Amol/l SOD, (o) 0.1 Amol/l SOD + 0.1 mmol/l NADH. All other conditions are as in Fig. 1.
not influence the autoxidation rate of HbO2, but it nullifies the effect due to the presence of 0.1 mmol/l NADH. The autoxidation of HbA in the presence of catalase (Fig. 3) drops below the control level both in the presence and in the absence of NADH. SOD and
CAT together have a stronger protective effect than any of these enzymes individually (data not shown). In the same experimental conditions, NADH is oxidized in the presence of hemoglobin (Fig. 4). SOD, CAT and both these enzymes together decrease
Fig. 3. Inhibition of met-Hb formation rate by catalase (0.1 Amol/l) in the presence and in the absence of NADH: (*) control, (E) 0.1 mmol/l NADH, (.) 0.1 Amol/l CAT, (o) 0.1 Amol/l CAT + 0.1 mmol/l NADH. All other conditions are as in Fig. 1.
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Fig. 4. Rate of NADH oxidation in the presence of hemoglobin and antioxidant enzymes: (.) 0.1 mmol/l NADH (5) + 0.1 Amol/l SOD, (*) + 0.1 Amol/l CAT, (E) + 0.1 Amol/l SOD and 0.1 Amol/l CAT. All other conditions are as in Fig. 1.
the rate of NADH oxidation (SOD < CAT V SOD and CAT). The effect of NADH on met-Hb formation at pH 6.8 was also studied. The increased rate of autoxidation at
lower pH is further enhanced in the presence of NADH (Fig. 5). After 3 h of incubation in this condition, metHb formation reaches 11% in the control and almost 25% in the presence of 0.1 mmol/l NADH. However,
Fig. 5. Effect of superoxide dismutase and catalase on the rate of met-Hb formation in the presence and in the absence of 0.1 mmol/l NADH : (*) control, (x) 0.1 mmol/l NADH, (E) 0.1 Amol/l SOD, (D) 0.1 Amol/l SOD + 0.1 mmol/l NADH (.) 0.1 Amol/l CAT, (o) 0.1 Amol/l CAT + 0.1 mmol/l NADH. Hemoglobin (1 mg/ml) was incubated in 50 mmol/l Bis-Tris + 0.1 mmol/l EDTA pH. 6.8 at 35 jC.
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Fig. 6. Influence of hemoglobin and antioxidant enzymes on NADH oxidation: (.) 0.1 mmol/l NADH (5) + 0.1 Amol/l: SOD, (*) + 0.1 Amol/l CAT, (E) + 0.1 Amol/l SOD and 0.1 Amol/l CAT. All other conditions are as in Fig. 5.
the protective effect of SOD is much stronger at this pH and comparable to the effect of CAT (Fig. 5). In addition, the rate of NADH oxidation at pH 6.8 is increased. During the first 240 min at this pH (Fig. 6), the rate is about three times higher than at pH 7.4. The protective effect of SOD is also stronger at this pH than at pH 7.4.
4. Discussion This study was designed to answer the question of whether or not NADH can influence the process of HbA autoxidation. This coenzyme can reduce met-Hb to its oxy-form in the presence of methemoglobin reductase or electron carriers [4,5]. However, it can also act as an electron donor for some reactive oxygen species at relatively high rate. It has been reported that thiol radicals react directly with NADH and the rate constants have been determined to be 5.8 108 M 1 s 1 for cysteine and 2.3 108 M 1 s 1 for glutathione [10,11]. NADH also reacts with superoxide anion radical (Eq. (1)) and the reaction rate is much higher when NADH is bound to the enzymes lactate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase [6,12]. This is probably associated with
changes in NADH conformation [13]. Hemoglobin too, can bind reduced pyridine nucleotides [14] but it has not been proven whether this binding might induce the same conformational changes in the pyridine nucleotides. The data obtained clearly indicate that NADH alone accelerates met-Hb formation and this effect increases at lower pH rates. Destabilization of hemoglobin is accompanied by the oxidation of the cofactor. The presence of superoxide dismutase does not influence (in our experimental conditions) the HbA autoxidation rate at pH 7.4, but it decreases the rate at pH 6.8. Increasing the amount of superoxide anion radical produced at pH 6.8 with respect to pH 7.4, as well as a higher concentration of H + may accelerate their reaction with NADH (Eq. (1)). Due to this reaction, hydrogen peroxide and NAD radical are formed. NAD radical can react with O2 generating another superoxide radical (Eq. (2)). Thus, NADH oxidation by superoxide formed from autoxidation of hemoglobin will be accompanied by an increased formation of reactive oxygen species such as hydrogen peroxide and superoxide anion radical. Our data support this assumption since stimulation of Hb autoxidation in the presence of NADH and the pro-
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R.A. Olek et al. / Clinica Chimica Acta 324 (2002) 129–134
tective effect of both superoxide dismutase and catalase was observed. These data can be criticized because they are highly unlikely to occur in in vivo conditions due to the very low concentration of NADH in erythrocytes, which is less than 1:50 compared to the hemoglobin tetramer [15]. However, it should be taken into account that there are several enzymatic reactions that can regenerate NADH from NAD. Therefore, the turnover of NADH could stimulate the formation of free radicals due to reaction with oxy-hemoglobin. The results reported here may be further supported by the similar effect observed upon interaction of NADH with myoglobin (unpublished data). In this case, the formation of free radicals could have a more physiological importance, given that in the muscle the ratio between NADH and hemoproteins is higher with respect to erythrocytes.
References [1] Misra HP, Fridovich I. The generation of superoxide radical during the autoxidation of hemoglobin. J Biol Chem 1972; 247:6960 – 2. [2] Winterbourn CC, McGrath BM, Carrell RW. Reactions involving superoxide and normal and unstable haemoglobins. Biochem J 1976;155:493 – 502. [3] Paul WD, Kemp CR. Methemoglobin: a normal constituent of blood. Proc Soc Exp Biol 1944;56:55 – 6. [4] Kajita A, Noguchi K, Shukuya R. A simple non-enzymatic method to regenerate oxyhemoglobin from methemoglobin. Biochem Biophys Res Commun 1970;39:1199 – 204.
[5] Brown WD, Snyder HE. Nonenzymatic reduction and oxidation of myoglobin and hemoglobin by nicotinamide adenine dinucleotides and flavins. J Biol Chem 1969;244:6702 – 6. [6] Bielski BHJ, Chan PC. Kinetic study by pulse radiolysis of the lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by HO2 and O2 radicals. J Biol Chem 1974;250:318 – 21. [7] Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 1995;18(2):321 – 36. [8] Persson HL, Svensson AI, Brunk UT. A-lipoic acid and alipoamide prevent oxidant-induced lysosomal rupture and apoptosis. Redox rep 2001;6(5):327 – 34. [9] Rossi-Fanelli A, Antonini E, Caputo A. Studies on the relations between molecular and functional properties of hemoglobin. J Biol Chem 1961;236:391 – 6. [10] Forni LG, Willson RL. Thiyl and phenoxyl free radicals and NADH. Direct observation of one-electron oxidation. Biochem J 1986;240:897 – 903. [11] Forni LG, Willson RL. Thiyl free radicals and the oxidation of ferrocytochrome c. Direct observation of coupled hydrogen-atom- and electron-transfer reactions. Biochem J 1986; 240:905 – 7. [12] Chan PC, Bielski BHJ. Glyceraldehyde-3-phosphate dehydrogenase-catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals. J Biol Chem 1980; 255:874 – 6. [13] Meyer WL, Mahler HR, Baker RH. Nuclear magnetic resonance spectra and conformation of 1,4-dihydropyridines. Biochim Biophys Acta 1962;64:353 – 8. [14] Cashon R, Bonaventura C, Bonaventura J, Focesi A. The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin. J Biol Chem 1986;261:12700 – 5. [15] Gross RT, Schroeder EA, Gabrio BW. Pyridine nucleotides in erythrocyte metabolism. J Clin Invest 1966;45:249 – 55. .
Effect of NADH on the redox state of human hemoglobin Robert A. Olek a, Jedrzej Antosiewicz a, Gian Carlo Caulini b, Giancarlo Falcioni b,* a
Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education Wiejska 1, 80-336 Gdansk, Poland b Department of Biology MCA, University of Camerino, Via Camerini 2, I-62032 Camerino, Italy Received 6 February 2002; received in revised form 20 June 2002; accepted 27 June 2002
Abstract In this work, we report that NADH can increase the autoxidation rate of hemoglobin (HbA) in a pH-dependent fashion. During this process, this cofactor is itself oxidized. The presence of superoxide dismutase (SOD) and/or catalase (CAT) can inhibit this result. At lower pH rates, the effect of NADH on the hemoglobin autoxidation rate is more enhanced; in addition, the rate of NADH oxidation is increased. Our data indicates that the reduced pyridine nucleotide may influence the redox state of human hemoglobin by a mechanism, which probably involves free radical species. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Hemoglobin; NADH; Catalase; Superoxide dismutase; ROS
1. Introduction The physiological function of hemoglobin is to transport oxygen to the tissues, and this depends on the ability of the ferrous form (Hb2 + ) to reversibly bind molecular oxygen. However, oxy-hemoglobin can turn to met-Hb (the Hb3 + form), which is unable to transport oxygen. This conversion is associated with superoxide anion production [1] and thereby with products such as hydrogen peroxide or hydroxyl radicals, which can derive from superoxide anion itself. The superoxide anion radical and hydrogen peroxide can further react with hemoglobin, contributing to the level of methemoglobin produced [2].
*
Corresponding author. Tel.: +39-737-403211; fax: +39-737636216. E-mail address: [email protected] (G. Falcioni).
About 3% of met-Hb is produced in the erythrocyte in 24 h [3], but its level is maintained constantly low (about 1%) by the presence of met-hemoglobin reductases—NADH and NADPH-dependent enzymes. It has been reported that reduced pyridine nucleotides are also capable of non-enzymatic reduction of metHb but require the presence of an electron carrier [4] or EDTA [5]. On the other hand, it is well established that NADH can be oxidized by reactive oxygen species [6].
:
NADH þ O2 þ Hþ ! NAD: þ H2 O2
:
NAD: þ O2 ! NAD þ O2
ð1Þ ð2Þ
In this study, we have investigated the influence of NADH on the redox state of HbA and the role of SOD and CAT in this process, which probably involves reactive oxygen species.
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 2 4 2 - 5
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R.A. Olek et al. / Clinica Chimica Acta 324 (2002) 129–134
Experiments were carried out in the presence of different chelating agents such as EDTA, diethylenetriaminopentaacetic acid (DTPA) and deferoxamine, which as metal chelates are involved in the formation and quenching of reactive oxygen species [7,8].
2. Materials and methods Human hemoglobin (HbA) was obtained as previously described [9]. Hemoglobin at a concentration of 1 mg/ml was incubated in 10 mmol/l Tris – HCl buffer, pH 7.4, or 50 mmol/l bis-Tris buffer pH 6.8 at 35 jC. Incubation mixtures contained EDTA at a final concentration of 0.1 mmol/l. Autoxidation was monitored in a Cary 219 spectrophotometer, equipped with a thermostatically controlled cell holder. Changes in the absorption spectrum ranging from 500 to 700 nm were recorded as a function of time. Reference value for complete oxidation was estimated by addition of potassium ferricyanide. NADH oxidation was monitored spectrophotometrically at 340 nm. All the compounds, NADH as well as the enzymes superoxide dismutase (SOD; EC 1.15.1.1)
and catalase (CAT; EC 1.11.1.6) were purchased from Sigma-Aldrich.
3. Results The effect of increasing the concentration of NADH on the kinetics of HbO2 autoxidation was investigated. The time course of hemoglobin oxidation during incubation at 35 jC and pH 7.4 is reported in Fig. 1. It appears that NADH has a concentration-dependent accelerating effect on autoxidation. It is well to point out that 0.1 mmol/l EDTA was present in the medium for chelating free metal ions. After 3 h of incubation, met-Hb reaches 5.5% in the control and almost 20% in the presence of 0.5 mmol/l NADH. The effect of NADH in accelerating met-hemoglobin formation rate was present also when we used other chelating agents such as deferoxamine and DTPA (data not shown). NAD + has no effect on the autoxidation rate (data not shown). In order to assess a possible involvement of reactive oxygen species in this process, the effect of superoxide dismutase and catalase was investigated. The results reported in Fig. 2 show that the presence of 0.1 Amol/l SOD in our experimental conditions does
Fig. 1. Effect of NADH on the time course of hemoglobin autoxidation: (*) control, (E) 0.1 mmol/l NADH, (n) 0.3 mmol/l NADH, (.) 0.5 mmol/l NADH. Hemoglobin (1 mg/ml) was incubated in 10 mmol/l Tris – HCl + 0.1 mmol/l EDTA pH 7.4 at 35 jC.
R.A. Olek et al. / Clinica Chimica Acta 324 (2002) 129–134
131
Fig. 2. Influence of superoxide dismutase (0.1 AM) on the rate of met-Hb formation in the presence and in the absence of NADH: (*) control, (E) 0.1 mmol/l NADH, (.) 0.1 Amol/l SOD, (o) 0.1 Amol/l SOD + 0.1 mmol/l NADH. All other conditions are as in Fig. 1.
not influence the autoxidation rate of HbO2, but it nullifies the effect due to the presence of 0.1 mmol/l NADH. The autoxidation of HbA in the presence of catalase (Fig. 3) drops below the control level both in the presence and in the absence of NADH. SOD and
CAT together have a stronger protective effect than any of these enzymes individually (data not shown). In the same experimental conditions, NADH is oxidized in the presence of hemoglobin (Fig. 4). SOD, CAT and both these enzymes together decrease
Fig. 3. Inhibition of met-Hb formation rate by catalase (0.1 Amol/l) in the presence and in the absence of NADH: (*) control, (E) 0.1 mmol/l NADH, (.) 0.1 Amol/l CAT, (o) 0.1 Amol/l CAT + 0.1 mmol/l NADH. All other conditions are as in Fig. 1.
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Fig. 4. Rate of NADH oxidation in the presence of hemoglobin and antioxidant enzymes: (.) 0.1 mmol/l NADH (5) + 0.1 Amol/l SOD, (*) + 0.1 Amol/l CAT, (E) + 0.1 Amol/l SOD and 0.1 Amol/l CAT. All other conditions are as in Fig. 1.
the rate of NADH oxidation (SOD < CAT V SOD and CAT). The effect of NADH on met-Hb formation at pH 6.8 was also studied. The increased rate of autoxidation at
lower pH is further enhanced in the presence of NADH (Fig. 5). After 3 h of incubation in this condition, metHb formation reaches 11% in the control and almost 25% in the presence of 0.1 mmol/l NADH. However,
Fig. 5. Effect of superoxide dismutase and catalase on the rate of met-Hb formation in the presence and in the absence of 0.1 mmol/l NADH : (*) control, (x) 0.1 mmol/l NADH, (E) 0.1 Amol/l SOD, (D) 0.1 Amol/l SOD + 0.1 mmol/l NADH (.) 0.1 Amol/l CAT, (o) 0.1 Amol/l CAT + 0.1 mmol/l NADH. Hemoglobin (1 mg/ml) was incubated in 50 mmol/l Bis-Tris + 0.1 mmol/l EDTA pH. 6.8 at 35 jC.
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133
Fig. 6. Influence of hemoglobin and antioxidant enzymes on NADH oxidation: (.) 0.1 mmol/l NADH (5) + 0.1 Amol/l: SOD, (*) + 0.1 Amol/l CAT, (E) + 0.1 Amol/l SOD and 0.1 Amol/l CAT. All other conditions are as in Fig. 5.
the protective effect of SOD is much stronger at this pH and comparable to the effect of CAT (Fig. 5). In addition, the rate of NADH oxidation at pH 6.8 is increased. During the first 240 min at this pH (Fig. 6), the rate is about three times higher than at pH 7.4. The protective effect of SOD is also stronger at this pH than at pH 7.4.
4. Discussion This study was designed to answer the question of whether or not NADH can influence the process of HbA autoxidation. This coenzyme can reduce met-Hb to its oxy-form in the presence of methemoglobin reductase or electron carriers [4,5]. However, it can also act as an electron donor for some reactive oxygen species at relatively high rate. It has been reported that thiol radicals react directly with NADH and the rate constants have been determined to be 5.8 108 M 1 s 1 for cysteine and 2.3 108 M 1 s 1 for glutathione [10,11]. NADH also reacts with superoxide anion radical (Eq. (1)) and the reaction rate is much higher when NADH is bound to the enzymes lactate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase [6,12]. This is probably associated with
changes in NADH conformation [13]. Hemoglobin too, can bind reduced pyridine nucleotides [14] but it has not been proven whether this binding might induce the same conformational changes in the pyridine nucleotides. The data obtained clearly indicate that NADH alone accelerates met-Hb formation and this effect increases at lower pH rates. Destabilization of hemoglobin is accompanied by the oxidation of the cofactor. The presence of superoxide dismutase does not influence (in our experimental conditions) the HbA autoxidation rate at pH 7.4, but it decreases the rate at pH 6.8. Increasing the amount of superoxide anion radical produced at pH 6.8 with respect to pH 7.4, as well as a higher concentration of H + may accelerate their reaction with NADH (Eq. (1)). Due to this reaction, hydrogen peroxide and NAD radical are formed. NAD radical can react with O2 generating another superoxide radical (Eq. (2)). Thus, NADH oxidation by superoxide formed from autoxidation of hemoglobin will be accompanied by an increased formation of reactive oxygen species such as hydrogen peroxide and superoxide anion radical. Our data support this assumption since stimulation of Hb autoxidation in the presence of NADH and the pro-
134
R.A. Olek et al. / Clinica Chimica Acta 324 (2002) 129–134
tective effect of both superoxide dismutase and catalase was observed. These data can be criticized because they are highly unlikely to occur in in vivo conditions due to the very low concentration of NADH in erythrocytes, which is less than 1:50 compared to the hemoglobin tetramer [15]. However, it should be taken into account that there are several enzymatic reactions that can regenerate NADH from NAD. Therefore, the turnover of NADH could stimulate the formation of free radicals due to reaction with oxy-hemoglobin. The results reported here may be further supported by the similar effect observed upon interaction of NADH with myoglobin (unpublished data). In this case, the formation of free radicals could have a more physiological importance, given that in the muscle the ratio between NADH and hemoproteins is higher with respect to erythrocytes.
References [1] Misra HP, Fridovich I. The generation of superoxide radical during the autoxidation of hemoglobin. J Biol Chem 1972; 247:6960 – 2. [2] Winterbourn CC, McGrath BM, Carrell RW. Reactions involving superoxide and normal and unstable haemoglobins. Biochem J 1976;155:493 – 502. [3] Paul WD, Kemp CR. Methemoglobin: a normal constituent of blood. Proc Soc Exp Biol 1944;56:55 – 6. [4] Kajita A, Noguchi K, Shukuya R. A simple non-enzymatic method to regenerate oxyhemoglobin from methemoglobin. Biochem Biophys Res Commun 1970;39:1199 – 204.
[5] Brown WD, Snyder HE. Nonenzymatic reduction and oxidation of myoglobin and hemoglobin by nicotinamide adenine dinucleotides and flavins. J Biol Chem 1969;244:6702 – 6. [6] Bielski BHJ, Chan PC. Kinetic study by pulse radiolysis of the lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by HO2 and O2 radicals. J Biol Chem 1974;250:318 – 21. [7] Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 1995;18(2):321 – 36. [8] Persson HL, Svensson AI, Brunk UT. A-lipoic acid and alipoamide prevent oxidant-induced lysosomal rupture and apoptosis. Redox rep 2001;6(5):327 – 34. [9] Rossi-Fanelli A, Antonini E, Caputo A. Studies on the relations between molecular and functional properties of hemoglobin. J Biol Chem 1961;236:391 – 6. [10] Forni LG, Willson RL. Thiyl and phenoxyl free radicals and NADH. Direct observation of one-electron oxidation. Biochem J 1986;240:897 – 903. [11] Forni LG, Willson RL. Thiyl free radicals and the oxidation of ferrocytochrome c. Direct observation of coupled hydrogen-atom- and electron-transfer reactions. Biochem J 1986; 240:905 – 7. [12] Chan PC, Bielski BHJ. Glyceraldehyde-3-phosphate dehydrogenase-catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals. J Biol Chem 1980; 255:874 – 6. [13] Meyer WL, Mahler HR, Baker RH. Nuclear magnetic resonance spectra and conformation of 1,4-dihydropyridines. Biochim Biophys Acta 1962;64:353 – 8. [14] Cashon R, Bonaventura C, Bonaventura J, Focesi A. The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin. J Biol Chem 1986;261:12700 – 5. [15] Gross RT, Schroeder EA, Gabrio BW. Pyridine nucleotides in erythrocyte metabolism. J Clin Invest 1966;45:249 – 55. .