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Stimulatory effect of ceruloplasmin on Fe2+ induced oxidation of oxyhemoglobin
Int. J. Biochem. Vol. 14, pp. 1043to 1046,1982 Printed in Great Britain. All rights reserved
STIMULATORY INDUCED
0020-711X/82/121043-04$03.00/0 Copyright 0 1982Pergamon Press Ltd
EFFECT OF CERULOPLASMIN ON Fe2+ OXIDATION OF OXYHEMOGLOBIN
ROLF A. L~VSTAD Institute of Medical Biochemistry, University of Oslo, Sognsvannsveien 9, Oslo 3, Norway (Received
17 March 1982)
Abstract-l.
Fe’+ ion dependent oxidation of oxyhemoglobin to methemoglobin is markedly enhanced by human ceruloplasmin. 2. It is suggested that the effect is due to an interaction between a ceruloplasmin-iron complex and oxyhemoglobin.
INTRODUCTION
The rate of oxyhemoglobin oxidation to methemoglobin is affected by various factors (K&e, 1974), including several metal ions. The effect of copper ions has been investigated by Rifiind (1974) and Winterbourn & Carrel1 (1977), while Kikugawa et al. (1981) studied the stimulatory effect of iron ions on the oxyhemoglobin oxidation rate. Fe*+ ions readily bind to ceruloplasmin (ferroxidase, EC 1.16.3. l), a blue copper containing serum protein, which rapidly oxidizes Fe” ions to Fe3+ in a catalytic manner (Curzon & O’Reilly, 1960; Osaki et al., 1966; McDermott et al., 1968); the process transforming molecular oxygen to water. Ceruloplasmin contains six or seven firmly bound copper ions per molecule, the metal ions being essential for the oxidase activity of the protein (Frieden & Hsieh, 1976; RydCn & Bjark, 1976). The present communication shows that low concentrations of ceruloplasmin markedly enhance the Fe’+ ion induced oxidation of oxyhemoglobin. EXPERIMENTAL
Materials
Human ceruloplasmin was obtained from AB Kabi. The protein solution was dialyzed against 0.1 M sodium acetate buffer, pH 5.6, in order to remove contaminating copper ions, usually associated with cerulopl,asmin preparations (Lsvstad, 1979). The ceruloplasmin solution had an absorbance ratio, A610n,,JAZSOnm, of 0.033. Enzyme concentrations were calculated from the 610nm absorption (E = 10.9 mM- 1cm- ‘) (Deutsch, 1960).
Suoeroxide dismutase (EC 1.15.1.1) and catalase (EC 1.1 l.i.6) were purchased irom Sigma’Chemical Co., gnd (NH&Fe(SO&, FeCl, and NaN, (sodium azide) from E. Merck AG. All aqueous solutions were made in deionized, glass-distilled water. Iron salts were dissolved in water immediately before use. Superoxide dismutase and catalase were routinely added to oxyhemoglobin containing reaction solutions in order to prevent a possible accumulation of superoxide radicals and hydrogen peroxide, respectively. The latter compounds are both reported to oxidize oxyhemoglobin to methemoglobin (Winterbourn et al., 1976). Preparation Human
of hemoglobin erythrocytes
were washed
five times with lo-v01 1043
portions of cold phosphate-saline buffer (155 mM NaCl in 10 mM sodium phosphate buffer, pH 7.4). The erythrocytes were then hemolyzed with a lo-v01 portion of cold, deionized water, and the preparation was centrifuged for 30 min at 27,000 y. A 15 ml sample of the supernatant was applied on a DEAE-Sephadex A-SO column (2.5 x 25 cm), previously equilibrated with 10 mM sodium phosphate buffer, pH 7.4. The hemoglobin solution then obtained was dialyzed against 10 mM sodium phosphate buffer, pH 7.4, in the cold room (4°C). Apparatus A Pye-Unicam SP8-250 instrument photometric measurements.
was used for spectro-
RESULTS Figure la shows that when 0.5 mM Fe’+ ions are added to a ceruloplasmin containing solution of oxyhemoglobin, a rapid decrease in the absorbance at 577 nm (A A,) of oxyhemoglobin is observed. The concentration of ceruloplasmin used only slightly affected the stability of oxyhemoglobin. When Fe’+ ions are added in the absence of ceruloplasmin the initial rapid decrease does not take place (Fig. la). Analysis of the visible spectrum of the reaction solution, after the ceruloplasmin dependent initial decrease of the 577 nm absorbance is over, shows that also the 541 nm absorption band of oxyhemoglobin is decreased, while new absorption bands with maxima around 500 and 630 nm appear (Fig. lb). Furthermore, the Soret band is shifted from 414 nm to lower wavelengths. The spectral changes suggest that methemoglobin is formed during the process, and that the reaction solution contains a mixture of oxyhemoglobin and methemoglobin. The effect of ceruloplasmin concentration on the rate of Fe’+ ion dependent initial oxidation of oxyhemoglobin is shown in Fig. 2; the rate, v being calculated from the decrease in the 577 nm absorbance of oxyhemoglobin (q,_ = 14.6 mM- ’ cm- ‘) (Antonini & Brunori, 1975). The rate was found to be linearly dependent on the ceruloplasmin concentration. Figure 2 also shows that the amount of oxyhemoglobin oxidized during the initial, rapid reaction, calculated from A A, (Fig. la), is dependent on the concentration of the enzyme.
1044
ROLF A. LGWSTAD
b
3 Fe2+
I
I
I
4
6
500
Time,
600
min
700
nm
Fig. l.(a) Time course of Fe’+ ran induced oxidation of oxyhemoglobin in the absence (------) and presence (---) of ceruloplasmin. The reaction solution contained 17 PM oxyhemoglobin, 0.8 PM ceruloplasmin, 10 pg/ml superoxide dismutase, 10 pg/ml catalase, 1 mM sodium phosphate and 0.5 mM Fez+ ions in 0.125 M sodium acetate buffer, pH 5.6 (37°C). (b) Visible spectrum of the ceruloplasmin containing reaction solution recorded before (- - - -- -) and after (---) the Fe ‘+ ion induced rapid decrease of the 577 nm absorbance.
Reaction rates and amounts of hemoglobin oxidized initially were also calculated from the time course of methemoglobin formation at 630nm. The data obtained were in excellent agreement with those shown in Fig. 2. Figure 3 shows that the initial oxidation rate, v is also linearly dependent on the concentration of oxyhemoglobin, when the ceruloplasminand Fe’+ ion concentrations were kept constant. The effect of different concentrations of iron ions
on the oxidation rate of oxyhemoglobin is shown in Fig. 4. The initial rate obtained by addition of Fe’+ ions in the presence of ceruloplasmin was found to be constant in the Fe” ion concentration range investigated. Addition of Fe3+ ions instead of Fe2+ ions had little effect on the oxidation rate (Fig. 4), both in the absence or presence of ceruloplasmin. The effect of azide on the ceruloplasmin-Fe2+ dependent oxidation was also investigated. Azide is a powerful inhibitor of ceruloplasmin oxidase activity
.
IO -
. .E E
2
./
I
2
5-
H i
.
1’
0
0.6
0.4 Ceruloplasmin,
PM
L 0
/
J
.
, IO
20
oxyHb,
Fig. 2. Effect of ceruloplasmin concentration on the initial, Fe” ion induced rate of oxyhemoglobin oxidation (-), and on the amount of oxyhemoglobin rapidly oxidized initially (------). The reaction solutions contained 15 PM oxyhemoglobin, ceruloplasmin (0.125-0.8 PM), 10 pg/ml superoxide dismutase, 10 pg/ml catalase. 1 mM sodium phosphate and 0.5 mM Fe ‘+ ions in 0.125 M sodium acetate buffer, pH 5.6 (37°C).
I
30
pM
Fig. 3. Effect of oxyhemoglobin concentration on the ceruloplasmin-Fe” ion dependent rate of oxyhemoglobin oxidation. The reaction solutions contained oxyhemoglobin (6.5532 PM), 1.0 PM ceruloplasmin, 10 &ml superoxide dismutase, 10 &ml catalase. 1 mM sodium phosphate and 0.5 mM Fe’+ ions in 0.125 M sodium acetate buffer, pH 5.6 (37°C).
Oxidation of hemoglobin by Fe”
,s
‘\
5.0
E
lr I. !
.
l-07 .
0.5
0.25
0
Fe2+or
Fe”+ f
mM
Fig. 4. Effect of Fe2+ ion- and Fe3+ ion concentrations on the rate of oxyhemoglobin oxidation in the absence and presence of ceruloplasmin. The reaction solutions contained 15 PM oxyhemoglobin, 1.0 PM ceruloplas~n, 10 pg/mJ superoxide dismutase, 10 pg/ml catalase, 1 mM sodium phosphate and iron ions (0.1-0.5 mM) in 0.125 M sodium acetate buffer, pH 5.6 (37°C). l, Fe”. ions + ceruions; A, Fe3* ions + ceruloplasmin; y;;as,; 0, Fe” ions; x, Fez+ ions i ceruloplasmin and 1 mM azide.
(Curzon, 19663,and as shown in Fig. 4, it completely prevented the rapid oxidation of oxyhemoglobin.
1045
between ceruloplasmin-iron complex and oxyhemoglobin. From Fig. 2 a pseudo first order rate constant, k’ = V/[CP], is calculated ([CP] = cernloplasmin concentration). In this case [CP] is also equal to the ~ruloplasmin-iron complex concentration, since the enzyme is saturated with iron ions during the experiment (Osaki, 1966). The second order rate constant, k, is equal to k’/[oxyHb]. The values obtained for k’ and k are 4.9 min - ’ and 0.33 ,uM-’ min- ‘, respectively. Figure 4 shows that in the absence of ceruloplasmin Fe” ions slowly oxidize oxyhemoglobin, as previously reported by Kikugawa et al. (1981). FeZi ions are unstable, being readily oxidized to Fe3+ ions within a short time (Goto et al., 1970; Levstad, 1981); thus the oxyhemoglobin oxidation may in reality be due to electron accepting Fe3’ ions. Oxygen molecules are reduced by Fe2+ ions to superoxide radicals (Goto et al., 1970), which also oxidize oxyhemoglobin ~interbo~rn et al., 1976). In the present experiments superoxide dismutase, a superoxide radical scavenger, is added to reaction solutions, preventing the latter reaction. ~ckn~w~e~ge~e~r~.The financial support from Professor Einar Langfeldts fond is gratefully acknowledged. The author thanks Mrs Bjerg Tonsberg for skilfuf technical assistance.
DISCUSSION
The stimulatory effect of ceruloplasmin on Fe” ion induced oxyhemoglobin oxidation is probably related to the ferroxidase activity of the protein, since azide, an effective ceruloplasmin inhibitor (Curzon, 1966), eliminates the effect of the enzyme (Fig. 4). Without azide the oxyhemoglobin oxidation rate is linearly dependent on enzyme concentration (Fig. 2), but independent of the concentration of Fez+ ions (Fig. 4). The latter observation is probably due to the fact that all ceruloplasmin molecules are bound to iron ions in the Fezi ion concen~ation range used. Ceruloplasmin has a high affinity for Fe2’ ions (Osaki, 1966; Osaki & Walaas, 1967), the ceruloplasmin-iron reaction system being characterized by low &-values (Osaki, 1966). The rapid removal of an electron from oxyhemoglobin could either be due to electron accepting Fe3” ions, generated by the action of ceruloplasmin on Fe”’ ions, or to the ceruloplasmin-iron complex itself. Experiments showed that Fe3+ ions had little effect on the oxyhemoglobin oxidation rate (Fig. 4), although prolonged incubation revealed that Fe3+ ions did increase the oxidation rate (Kikugawa et al., 1981). It is suggested that the rapid ceruloplasminFe ” ion dependent oxidation of oxyhemoglobin is caused by an interaction between the ceruloplasminiron complex and oxyhemoglobin, the complex effectively removing an electron from the oxyhemoglobin molecule. In this connection it is interesting that other iron ion complexing agents, such as EDTA and ADP, markedly stimulate the Fe” ion induced oxyhemoglobin oxidation (Kikugawa et al., 1981). The linear dependence of oxidation rate, V, on both ceruloplasmin- and oxyhemoglobin concentrations (Figs 2 and 3) suggests a second order reaction
REFERENCES ANTONINI
E. & BRUNORIM. (1975) Hemoglobin and methemoglobin. In The Red Blood Cell (Edited by SURGENOR D. M.) Vol II, pp. 753-797. Academic Press, New York. CLJRZON G. (1966) The inhibition of caeruloplasmin by azide. Biochem. .I. 100,295-302. CURZ~N G. & O’REILLYS. (1960) A coupled iron-caeruloplasmin oxidation system. Biochem. biophys. Res. Commun. 2,284-286. DEUTSCH N.
F. (1960) The preparation of crystalline ceruloplasmin from human plasma. Arclas Biachem. ~~op~ys. 89, 225--229. FRIEDENE. & HSIE~ H. S. (1976) Ceruloplasmin: The copper transport protein with essential oxidase activity. Adv.
Enzym. 44, 187-236. Goro K., TAMURAH. & NAGAYANIA M. (1970) The mech-
anism of oxidation of ferrous ion in neutral solution. Inorg. Chem. 9,963-964.
KIESE-M. (1974) Methemoglobinemia: A Comprehensive Treatise. CRC Press, Cleveland, Ohio. KIKIIGAWAK., SASAH~R~T., S&AKI T. & KURE~HI T. (1981) Factors in~uencing the autoxidation of hemoglobin A. Chem. pharm. EN. 29, 1382- 1389. L~VSTADR. A. (1979) Activating effect of copper ions on the interaction of ceruloplasmin with catecholamines, Gea. ~harmac. 10, 147-151. L~VSTADR. A. (1981) The protective action of cerufoplasmin on Fe’+ stimulated lysis of rat erythrocytes. Int. J. Biochem. 13, 221-224. MCDERMOTTJ. A., HUEER C. T., OSAKI S. & FRIEDENE. (1968) Role of iron in the oxidase activity of ceruloplasmin. ~iuch~~?z.biup~zys. Actu 151, 541--557. OSAKK S. (1966) Kinetic studies of ferrous ion oxidation with crystalline human ferroxidase (ceruloplasmin), I. J. bio/. Chem. 241, 5053-5059. OSAKI S. & WALAAS0. (1967) Kinetic studies of ferrous ion oxidation with crystalline human ferroxidase. II. J. biol. C/rem. 242, 2653-2657.
1046
ROLF A. L~VSTAD
OSAKI S., JOHNSON D. A. & FRIEDEN E. (1966) The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum. J. bid. Chem. 241, 2746-2751. RIFKIND J. M. (1974) Copper and the autoxidation of hemoglobin. Biochemisrr~ -13, 2475-2481. RYD~N L. & BJ~RK I. (1976) Reinvestigation of some physicochemical and chemical properties of human
ceruloplasmin (ferroxidase). Biochemistry 15, 341 l3417. WINTERBOURN C. C. & CARRELL R. W. (1977) Oxidation of human haemoglobin by copper. Biochern. .I. 165, 141-14s. WINTERBOURN C. C., MCGRATH B. M. & CARRELL R. M. (1976) Reactions involving superoxide and normal and unstable haemoglobins. Biochern. J. 155, 493-502.
STIMULATORY INDUCED
0020-711X/82/121043-04$03.00/0 Copyright 0 1982Pergamon Press Ltd
EFFECT OF CERULOPLASMIN ON Fe2+ OXIDATION OF OXYHEMOGLOBIN
ROLF A. L~VSTAD Institute of Medical Biochemistry, University of Oslo, Sognsvannsveien 9, Oslo 3, Norway (Received
17 March 1982)
Abstract-l.
Fe’+ ion dependent oxidation of oxyhemoglobin to methemoglobin is markedly enhanced by human ceruloplasmin. 2. It is suggested that the effect is due to an interaction between a ceruloplasmin-iron complex and oxyhemoglobin.
INTRODUCTION
The rate of oxyhemoglobin oxidation to methemoglobin is affected by various factors (K&e, 1974), including several metal ions. The effect of copper ions has been investigated by Rifiind (1974) and Winterbourn & Carrel1 (1977), while Kikugawa et al. (1981) studied the stimulatory effect of iron ions on the oxyhemoglobin oxidation rate. Fe*+ ions readily bind to ceruloplasmin (ferroxidase, EC 1.16.3. l), a blue copper containing serum protein, which rapidly oxidizes Fe” ions to Fe3+ in a catalytic manner (Curzon & O’Reilly, 1960; Osaki et al., 1966; McDermott et al., 1968); the process transforming molecular oxygen to water. Ceruloplasmin contains six or seven firmly bound copper ions per molecule, the metal ions being essential for the oxidase activity of the protein (Frieden & Hsieh, 1976; RydCn & Bjark, 1976). The present communication shows that low concentrations of ceruloplasmin markedly enhance the Fe’+ ion induced oxidation of oxyhemoglobin. EXPERIMENTAL
Materials
Human ceruloplasmin was obtained from AB Kabi. The protein solution was dialyzed against 0.1 M sodium acetate buffer, pH 5.6, in order to remove contaminating copper ions, usually associated with cerulopl,asmin preparations (Lsvstad, 1979). The ceruloplasmin solution had an absorbance ratio, A610n,,JAZSOnm, of 0.033. Enzyme concentrations were calculated from the 610nm absorption (E = 10.9 mM- 1cm- ‘) (Deutsch, 1960).
Suoeroxide dismutase (EC 1.15.1.1) and catalase (EC 1.1 l.i.6) were purchased irom Sigma’Chemical Co., gnd (NH&Fe(SO&, FeCl, and NaN, (sodium azide) from E. Merck AG. All aqueous solutions were made in deionized, glass-distilled water. Iron salts were dissolved in water immediately before use. Superoxide dismutase and catalase were routinely added to oxyhemoglobin containing reaction solutions in order to prevent a possible accumulation of superoxide radicals and hydrogen peroxide, respectively. The latter compounds are both reported to oxidize oxyhemoglobin to methemoglobin (Winterbourn et al., 1976). Preparation Human
of hemoglobin erythrocytes
were washed
five times with lo-v01 1043
portions of cold phosphate-saline buffer (155 mM NaCl in 10 mM sodium phosphate buffer, pH 7.4). The erythrocytes were then hemolyzed with a lo-v01 portion of cold, deionized water, and the preparation was centrifuged for 30 min at 27,000 y. A 15 ml sample of the supernatant was applied on a DEAE-Sephadex A-SO column (2.5 x 25 cm), previously equilibrated with 10 mM sodium phosphate buffer, pH 7.4. The hemoglobin solution then obtained was dialyzed against 10 mM sodium phosphate buffer, pH 7.4, in the cold room (4°C). Apparatus A Pye-Unicam SP8-250 instrument photometric measurements.
was used for spectro-
RESULTS Figure la shows that when 0.5 mM Fe’+ ions are added to a ceruloplasmin containing solution of oxyhemoglobin, a rapid decrease in the absorbance at 577 nm (A A,) of oxyhemoglobin is observed. The concentration of ceruloplasmin used only slightly affected the stability of oxyhemoglobin. When Fe’+ ions are added in the absence of ceruloplasmin the initial rapid decrease does not take place (Fig. la). Analysis of the visible spectrum of the reaction solution, after the ceruloplasmin dependent initial decrease of the 577 nm absorbance is over, shows that also the 541 nm absorption band of oxyhemoglobin is decreased, while new absorption bands with maxima around 500 and 630 nm appear (Fig. lb). Furthermore, the Soret band is shifted from 414 nm to lower wavelengths. The spectral changes suggest that methemoglobin is formed during the process, and that the reaction solution contains a mixture of oxyhemoglobin and methemoglobin. The effect of ceruloplasmin concentration on the rate of Fe’+ ion dependent initial oxidation of oxyhemoglobin is shown in Fig. 2; the rate, v being calculated from the decrease in the 577 nm absorbance of oxyhemoglobin (q,_ = 14.6 mM- ’ cm- ‘) (Antonini & Brunori, 1975). The rate was found to be linearly dependent on the ceruloplasmin concentration. Figure 2 also shows that the amount of oxyhemoglobin oxidized during the initial, rapid reaction, calculated from A A, (Fig. la), is dependent on the concentration of the enzyme.
1044
ROLF A. LGWSTAD
b
3 Fe2+
I
I
I
4
6
500
Time,
600
min
700
nm
Fig. l.(a) Time course of Fe’+ ran induced oxidation of oxyhemoglobin in the absence (------) and presence (---) of ceruloplasmin. The reaction solution contained 17 PM oxyhemoglobin, 0.8 PM ceruloplasmin, 10 pg/ml superoxide dismutase, 10 pg/ml catalase, 1 mM sodium phosphate and 0.5 mM Fez+ ions in 0.125 M sodium acetate buffer, pH 5.6 (37°C). (b) Visible spectrum of the ceruloplasmin containing reaction solution recorded before (- - - -- -) and after (---) the Fe ‘+ ion induced rapid decrease of the 577 nm absorbance.
Reaction rates and amounts of hemoglobin oxidized initially were also calculated from the time course of methemoglobin formation at 630nm. The data obtained were in excellent agreement with those shown in Fig. 2. Figure 3 shows that the initial oxidation rate, v is also linearly dependent on the concentration of oxyhemoglobin, when the ceruloplasminand Fe’+ ion concentrations were kept constant. The effect of different concentrations of iron ions
on the oxidation rate of oxyhemoglobin is shown in Fig. 4. The initial rate obtained by addition of Fe’+ ions in the presence of ceruloplasmin was found to be constant in the Fe” ion concentration range investigated. Addition of Fe3+ ions instead of Fe2+ ions had little effect on the oxidation rate (Fig. 4), both in the absence or presence of ceruloplasmin. The effect of azide on the ceruloplasmin-Fe2+ dependent oxidation was also investigated. Azide is a powerful inhibitor of ceruloplasmin oxidase activity
.
IO -
. .E E
2
./
I
2
5-
H i
.
1’
0
0.6
0.4 Ceruloplasmin,
PM
L 0
/
J
.
, IO
20
oxyHb,
Fig. 2. Effect of ceruloplasmin concentration on the initial, Fe” ion induced rate of oxyhemoglobin oxidation (-), and on the amount of oxyhemoglobin rapidly oxidized initially (------). The reaction solutions contained 15 PM oxyhemoglobin, ceruloplasmin (0.125-0.8 PM), 10 pg/ml superoxide dismutase, 10 pg/ml catalase. 1 mM sodium phosphate and 0.5 mM Fe ‘+ ions in 0.125 M sodium acetate buffer, pH 5.6 (37°C).
I
30
pM
Fig. 3. Effect of oxyhemoglobin concentration on the ceruloplasmin-Fe” ion dependent rate of oxyhemoglobin oxidation. The reaction solutions contained oxyhemoglobin (6.5532 PM), 1.0 PM ceruloplasmin, 10 &ml superoxide dismutase, 10 &ml catalase. 1 mM sodium phosphate and 0.5 mM Fe’+ ions in 0.125 M sodium acetate buffer, pH 5.6 (37°C).
Oxidation of hemoglobin by Fe”
,s
‘\
5.0
E
lr I. !
.
l-07 .
0.5
0.25
0
Fe2+or
Fe”+ f
mM
Fig. 4. Effect of Fe2+ ion- and Fe3+ ion concentrations on the rate of oxyhemoglobin oxidation in the absence and presence of ceruloplasmin. The reaction solutions contained 15 PM oxyhemoglobin, 1.0 PM ceruloplas~n, 10 pg/mJ superoxide dismutase, 10 pg/ml catalase, 1 mM sodium phosphate and iron ions (0.1-0.5 mM) in 0.125 M sodium acetate buffer, pH 5.6 (37°C). l, Fe”. ions + ceruions; A, Fe3* ions + ceruloplasmin; y;;as,; 0, Fe” ions; x, Fez+ ions i ceruloplasmin and 1 mM azide.
(Curzon, 19663,and as shown in Fig. 4, it completely prevented the rapid oxidation of oxyhemoglobin.
1045
between ceruloplasmin-iron complex and oxyhemoglobin. From Fig. 2 a pseudo first order rate constant, k’ = V/[CP], is calculated ([CP] = cernloplasmin concentration). In this case [CP] is also equal to the ~ruloplasmin-iron complex concentration, since the enzyme is saturated with iron ions during the experiment (Osaki, 1966). The second order rate constant, k, is equal to k’/[oxyHb]. The values obtained for k’ and k are 4.9 min - ’ and 0.33 ,uM-’ min- ‘, respectively. Figure 4 shows that in the absence of ceruloplasmin Fe” ions slowly oxidize oxyhemoglobin, as previously reported by Kikugawa et al. (1981). FeZi ions are unstable, being readily oxidized to Fe3+ ions within a short time (Goto et al., 1970; Levstad, 1981); thus the oxyhemoglobin oxidation may in reality be due to electron accepting Fe3’ ions. Oxygen molecules are reduced by Fe2+ ions to superoxide radicals (Goto et al., 1970), which also oxidize oxyhemoglobin ~interbo~rn et al., 1976). In the present experiments superoxide dismutase, a superoxide radical scavenger, is added to reaction solutions, preventing the latter reaction. ~ckn~w~e~ge~e~r~.The financial support from Professor Einar Langfeldts fond is gratefully acknowledged. The author thanks Mrs Bjerg Tonsberg for skilfuf technical assistance.
DISCUSSION
The stimulatory effect of ceruloplasmin on Fe” ion induced oxyhemoglobin oxidation is probably related to the ferroxidase activity of the protein, since azide, an effective ceruloplasmin inhibitor (Curzon, 1966), eliminates the effect of the enzyme (Fig. 4). Without azide the oxyhemoglobin oxidation rate is linearly dependent on enzyme concentration (Fig. 2), but independent of the concentration of Fez+ ions (Fig. 4). The latter observation is probably due to the fact that all ceruloplasmin molecules are bound to iron ions in the Fezi ion concen~ation range used. Ceruloplasmin has a high affinity for Fe2’ ions (Osaki, 1966; Osaki & Walaas, 1967), the ceruloplasmin-iron reaction system being characterized by low &-values (Osaki, 1966). The rapid removal of an electron from oxyhemoglobin could either be due to electron accepting Fe3” ions, generated by the action of ceruloplasmin on Fe”’ ions, or to the ceruloplasmin-iron complex itself. Experiments showed that Fe3+ ions had little effect on the oxyhemoglobin oxidation rate (Fig. 4), although prolonged incubation revealed that Fe3+ ions did increase the oxidation rate (Kikugawa et al., 1981). It is suggested that the rapid ceruloplasminFe ” ion dependent oxidation of oxyhemoglobin is caused by an interaction between the ceruloplasminiron complex and oxyhemoglobin, the complex effectively removing an electron from the oxyhemoglobin molecule. In this connection it is interesting that other iron ion complexing agents, such as EDTA and ADP, markedly stimulate the Fe” ion induced oxyhemoglobin oxidation (Kikugawa et al., 1981). The linear dependence of oxidation rate, V, on both ceruloplasmin- and oxyhemoglobin concentrations (Figs 2 and 3) suggests a second order reaction
REFERENCES ANTONINI
E. & BRUNORIM. (1975) Hemoglobin and methemoglobin. In The Red Blood Cell (Edited by SURGENOR D. M.) Vol II, pp. 753-797. Academic Press, New York. CLJRZON G. (1966) The inhibition of caeruloplasmin by azide. Biochem. .I. 100,295-302. CURZ~N G. & O’REILLYS. (1960) A coupled iron-caeruloplasmin oxidation system. Biochem. biophys. Res. Commun. 2,284-286. DEUTSCH N.
F. (1960) The preparation of crystalline ceruloplasmin from human plasma. Arclas Biachem. ~~op~ys. 89, 225--229. FRIEDENE. & HSIE~ H. S. (1976) Ceruloplasmin: The copper transport protein with essential oxidase activity. Adv.
Enzym. 44, 187-236. Goro K., TAMURAH. & NAGAYANIA M. (1970) The mech-
anism of oxidation of ferrous ion in neutral solution. Inorg. Chem. 9,963-964.
KIESE-M. (1974) Methemoglobinemia: A Comprehensive Treatise. CRC Press, Cleveland, Ohio. KIKIIGAWAK., SASAH~R~T., S&AKI T. & KURE~HI T. (1981) Factors in~uencing the autoxidation of hemoglobin A. Chem. pharm. EN. 29, 1382- 1389. L~VSTADR. A. (1979) Activating effect of copper ions on the interaction of ceruloplasmin with catecholamines, Gea. ~harmac. 10, 147-151. L~VSTADR. A. (1981) The protective action of cerufoplasmin on Fe’+ stimulated lysis of rat erythrocytes. Int. J. Biochem. 13, 221-224. MCDERMOTTJ. A., HUEER C. T., OSAKI S. & FRIEDENE. (1968) Role of iron in the oxidase activity of ceruloplasmin. ~iuch~~?z.biup~zys. Actu 151, 541--557. OSAKK S. (1966) Kinetic studies of ferrous ion oxidation with crystalline human ferroxidase (ceruloplasmin), I. J. bio/. Chem. 241, 5053-5059. OSAKI S. & WALAAS0. (1967) Kinetic studies of ferrous ion oxidation with crystalline human ferroxidase. II. J. biol. C/rem. 242, 2653-2657.
1046
ROLF A. L~VSTAD
OSAKI S., JOHNSON D. A. & FRIEDEN E. (1966) The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum. J. bid. Chem. 241, 2746-2751. RIFKIND J. M. (1974) Copper and the autoxidation of hemoglobin. Biochemisrr~ -13, 2475-2481. RYD~N L. & BJ~RK I. (1976) Reinvestigation of some physicochemical and chemical properties of human
ceruloplasmin (ferroxidase). Biochemistry 15, 341 l3417. WINTERBOURN C. C. & CARRELL R. W. (1977) Oxidation of human haemoglobin by copper. Biochern. .I. 165, 141-14s. WINTERBOURN C. C., MCGRATH B. M. & CARRELL R. M. (1976) Reactions involving superoxide and normal and unstable haemoglobins. Biochern. J. 155, 493-502.