- Email: [email protected]
Iron oxidation by casein
Vol.
182,
February
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
Pages
14, 1992
IRON OXIDATION T. Department
Received
COMMUNICATIONS
of
Chemistry
1047-1052
BY CASEIN
Emery
and Biochemistry, Utah Logan, Utah 84322-0300
State
University,
January 8, 1992
summary: Casein accelerates the oxidation of Fe(I1) to Fe(II1) and the resulting Fe(II1) remains strongly bound to the casein. Removal of phosphate from the casein abolishes the oxidative process. The oxidation rate is proportional to the casein concentration, and with high casein concentrations the rate is pseudo-first-order with respect to Fe(I1) with a half-life of approximately 2 minutes. The oxidized iron is stoichiometrically bound to the casein, each mg of casein binding approximately 10 fig of iron. The physiological significance is discussed. 0 1992Academic mess, Inc. It is transferrin microorganisms
well established that the iron-binding proteins, and egg ovotransferrin, can function to deprive of iron and thereby diminish the risk of infection Lactoferrin is a milk protein that bears a close (1, 2) structural homology to transferrin and is believed to play an important role in preventing gastrointestinal infection in nursing infants (3, 4). Lactoferrin diminishes the rate and extent of bacterial growth in human milk in vitro by forming a strong complex Although no known constituent of milk can compete with iron (5). thermodynamically with lactoferrin for Fe(III), it has been found that more iron in milk is associated with casein micelles than with lactoferrin (6). The affinity of lactoferrin for Fe(I1) is probably too weak to be of physiological significance. Consequently, if lactoferrin is to serve as an effective inhibitor of microbial growth in milk, it is necessary that the metal be in the trivalent oxidation state. The mechanism by which iron enters milk is unknown, but reductive iron transport mechanisms and cellular reductants (7) suggest that the entering metal may initially be in the divalent oxidation state, which would necessitate a mechanism for oxidation of the metal to the ferric state for subsequent binding by lactoferrin. casein, In this paper I report that the milk protein, can 0006-291X/92 $1.50 1047
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 182, No. 3, 1992 accelerate
resulting chemistry relevance
BIOCHEMICAL
the oxidation Fe(II1) remains of the process discussed.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Fe(I1) to Fe(II1) and that tightly complexed to the casein. is described and the physiological
the The
MATERIALS ARD METHODS a-Casein and dephosphorylated casein were obtained from Sigma Chemical Co. or isolated from fresh skimmed milk (8). All other Iron was assayed by the method of chemicals were reagent grade. Stookey without addition of ascorbate so that only Fe(I1) was Oxidation of Fe(I1) and binding of Fe(II1) product measured (9). to casein was followed by disappearance of Fe(I1) from the reaction A typical reaction mixture contained in a final volume of mixture. loo pg iron as ferrous ammonium sulfate, 1.0 mg casein, 10 ml: 0.05 M piperazine-N,N'-bis[2-ethanesulfonic] (PIPES) buffer, pH 6.5. The solution was maintained at 25OC and gently stirred by a slow stream of air or oxygen (1.2 ml/min) to replenish dissolved oxygen. Aliquots of 0.5 ml were removed and assayed for Fe(I1).
RESULTS Addition of ferrous salts to fresh whole milk results in a rapid oxidation of FeII) to Fe(II1). The oxidation can readily be visualized by the loss of ability of the metal to form colored complexes with Fe(I1) specific chelators or by color formation upon addition of Fe(II1) specific chelators. Xanthine oxidase, an enzyme found in high concentration in milk, can serve as a ferroxidase in animal tissues (lo), and it has been suggested that this enzyme might serve the same function in milk (7). However, oxidation of Fe(I1) by milk is not inhibited by 0.3 mM allopurinol, and neither xanthine oxidase nor lactoperoxidase exhibit any ferrous oxidase activity under my assay conditions (data not shown). Any substance that preferentially binds Fe(II1) to Fe(I1) can serve as a thermodynamic trap for the latter ion and, depending upon the oxidative mechanism, can potentially increase the rate of oxidation (11). The concentrations of casein in human and bovine milk have been reported to be 0.4 and 2.6 g/100 g, respectively 1 shows the acceleration of the oxidation of Fe(I1) (12) - Figure by a-casein at significantly lower concentrations. Dissolved serves as oxidant as demonstrated by the absence of oxygen oxidation when the reaction is carried out anaerobically. Neverthslesss, under my assay conditions the concentration of oxygen is not rate limiting since similar initial rates were 1048
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Minutes
Fioure 1. Effect of casein on iron oxidation. Disappearance of Fe(I1) from the reaction mixture is due to oxidation of the metal to Fe(II1). Reaction conditions are as described in Materials and Methods. Symbols are: (a), no casein; (a), partially dephosphorylated casein, 1.0 mg/ml; (m), casein, 0.5 rag/ml; (A), casein,
1.0 mg/ml;
(O), casein,
2.0 mghul.
observed whether stirring was accomplished by a stream of air or oxygen. Furthermore, Fe(I1) oxidation is dependent upon complexation of the Fe(II1) product by the phosphate groups of evidenced by casein as the observation that partially dephosphorylated casein shows a decreased oxidation rate (Fig. 1). Oxidation of Fe(I1) by casein can not strictly be referred to as a ferroxidase activity because the casein does not act in a catalytic manner. The casein is involved stoichiometrically. Titration of a solution of Fe(I1) with casein shows that approximately 10 pg of iron can be bound per mg of casein (Fig. 2). The oxidized metal is strongly bound to the protein and is not removed by extensive dialysis. The metal can be quantitatively recovered with the protein upon isoelectric precipitation at pH 4.6. Precise kinetic analysis of the casein-mediated oxidation of Fe(I1) is difficult because of the heterogenous nature of the protein and the difficulty in determining the precise number of phosphate groups involved in the reaction. It has been reported that aerobic binding of Fe(I1) to the phosphorylated proteins leads to oxidative destruction of phosphoserine residues accompanied by an increased absorption of ultraviolet light (13). I confirmed The casein mediated oxidation this observation (data not shown). of iron appears to be second order with respect to protein and Fe(I1). Figure 3 shows the pseudo-first-order kinetics of iron 1049
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
67B5 4. 32lOa
0 0.2
014
Milligrams
Ok
016
cash/ml
Ficure 2. Titration of Fe(X) with casein. Reactions conditions are as described in Materials and Methods except that for each increment of added casein the mixture was allowed to staud for 1 hr to ensure that maximum oxidation of Fe(I1) was attained. oxidation in the presence of a high concentration pseudo-first-order rate constant for Fe(I1) calculated by the method of Guggenheim (14) 0.3 min-l, corresponding to a half-life of Fe(I1) minutes.
of casein. The oxidation was and found to be oxidation of 2.4
DISCUSSION In their supplemented
efforts to find a suitable method of producing ironmilk, Hegenauer and co-workers observed that addition
Minutes
Fiaure 3. Initial rate of casein-mediated Fe(I1) oxidation. The reaction conditions are as described in Materials and Methods using casein at a concentration of 2 mg/ml. Insert: pseudofirst-order plot of the rate data by the method of Guggenheim (ref. 14). 1050
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of ferrous sulfate to bovine milk resulted in formation of Fe(III) bound to casein (6). They suggested that Fe(I1) first displaced Ca (II) from the protein with subsequent oxidation of the Fe(II)casein complex. My results support this suggestion. The rate of Fe(I1) oxidation is highly dependent upon the concentration of casein and the rate is pseudo-first-order with respect to Fe(I1) concentration at high concentrations of casein, ruling out a mechanism by which the metal is first oxidized by dissolved oxygen in a rate-limiting step. The observed increase in ultraviolet absorption and the observation that dephosphorylated casein is ineffective in accelerating the oxidation of Fe(I1) demonstrate that the phosphate content of this protein is not only of nutritional importance but may also serve an important function in regulating the oxidation state of iron. The amount of iron in milk varies from species to species with values ranging from 0.1-0.2 mg/l for cow's milk to 5-10 mg/l for rat's milk (15). A large variation among individuals has also been found in human milk, but it is generally agreed that the iron content is approximately 0.5 mg/l. The titration data of Figure 2 demonstrates that the amount of casein in milk of any species can rapidly oxidize and bind all of the iron present and can thus serve as a buffer to ensure that all of the metal is effectively bound to protein. The low concentration of iron in human milk has been a source of consternation to nutritionists fearful of the harmful consequences of iron deficiency in the newborn (16). Currently, the recommended daily allowance of iron for the newborn human is 10 mg which, in view of the actual amount found in human milk, would require iron supplementation for all breast-feeding infants. In contrast, many iron researchers believe that dietary iron is to be avoided in the newborn and serves only to increase the risk of gastro-intestinal tract infection, and that an important function of iron-binding proteins such as lactoferrin is to withhold iron from microorganisms (17). The results presented in this paper may support the latter hypothesis. The high casein concentration of milk ensures rapid oxidation of iron to the trivalent oxidation state with concomitant strong binding to the protein. The net effect make iron less available to intestinal may be to microorganisms as well as to hasten removal of the metal from the intestinal tract by more efficient absorption of the metal-protein complex. 1051
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS This Institute of Health. assistance
work was supported by grant AI-09580 from the National of Allergy and Infectious Diseases, National Institutes The author wishes to acknowledge the expert technical of Ms. Wei Zhang.
REFERENCES 1.
2. 3. 4. 5.
Puschmann, M., and Ganzoni, A.M. (1977) Infect. Immun. 17, 663-664. Alderton, G.W., Ward, V.A., and Fevold, H.L. (1946) Arch. Biochem. 11, 9-13. Arnold, R. R., Cole, M. F., and McGhee, J.R. (1977) Science 197, 263-265. Brock, J.H. (1980) Arch. Dis. Child. 55, 417-421. Griffiths, E., and Humphreys, J. (1977) Infect. Immun. 15, 396-401.
6. Hegenauer, J., Saltman, P., Ludwig, D., Ripley, L., and Ley, A. (1979) J. Agric. Food Chem. 27, 1294-1301. 7. Emery, T. (1987) in Iron Transport in Microbes, Plants and Animals (Winkelmann, G., van der Helm, D., and Neilands, J. B eds.) pp. 235-250, VCH, Weinheim, Federal Republic of G&nany. 8. Hollar, C.M., Law, A.J.R., Dalgleish, D.G., and Brown, R. (1991) J. Dairy Sci. 74, 2403-2409. 9. Stookey, L.L. (1970) Anal. Chem. 42, 779-781. 10. Topham, R-W., Walker, M.C., and Calisch, M.P. (1982) Biochem. Biophys. Res. Commun. 109, 1240-1246. 11. Thorstensen, K., and Aisen, P. (1990) Biochim. Biophys. Acta 1052, 29-35. 12. Jenness, R. (1988) in Fundamentals of Dairy Chemistry, 3d edition (Wong, N.P., ed.), pp. l-37, Van Nostrand Reinhold, NY. 13. Grant, C.T., and Taborsky, G. (1966) Biochemistry 5, 544-555. 14. Hammes, G.G. (1978), Principles of Chemical Kinetics, p. 9, Academic Press, New York. 15. Liinnerdal, B. (1990) in Iron Metabolism in Infants (Lonnerdal, B., Ed.) pp. 87-107, CRC Press, Boca Raton, FL. 16. Scrimshaw, N.S. (1991) Scientific American 265, 46-52. 17 Weinberg, &,. E.D. (1984) Physiol. Rev. 64, 65-102.
1052
182,
February
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
Pages
14, 1992
IRON OXIDATION T. Department
Received
COMMUNICATIONS
of
Chemistry
1047-1052
BY CASEIN
Emery
and Biochemistry, Utah Logan, Utah 84322-0300
State
University,
January 8, 1992
summary: Casein accelerates the oxidation of Fe(I1) to Fe(II1) and the resulting Fe(II1) remains strongly bound to the casein. Removal of phosphate from the casein abolishes the oxidative process. The oxidation rate is proportional to the casein concentration, and with high casein concentrations the rate is pseudo-first-order with respect to Fe(I1) with a half-life of approximately 2 minutes. The oxidized iron is stoichiometrically bound to the casein, each mg of casein binding approximately 10 fig of iron. The physiological significance is discussed. 0 1992Academic mess, Inc. It is transferrin microorganisms
well established that the iron-binding proteins, and egg ovotransferrin, can function to deprive of iron and thereby diminish the risk of infection Lactoferrin is a milk protein that bears a close (1, 2) structural homology to transferrin and is believed to play an important role in preventing gastrointestinal infection in nursing infants (3, 4). Lactoferrin diminishes the rate and extent of bacterial growth in human milk in vitro by forming a strong complex Although no known constituent of milk can compete with iron (5). thermodynamically with lactoferrin for Fe(III), it has been found that more iron in milk is associated with casein micelles than with lactoferrin (6). The affinity of lactoferrin for Fe(I1) is probably too weak to be of physiological significance. Consequently, if lactoferrin is to serve as an effective inhibitor of microbial growth in milk, it is necessary that the metal be in the trivalent oxidation state. The mechanism by which iron enters milk is unknown, but reductive iron transport mechanisms and cellular reductants (7) suggest that the entering metal may initially be in the divalent oxidation state, which would necessitate a mechanism for oxidation of the metal to the ferric state for subsequent binding by lactoferrin. casein, In this paper I report that the milk protein, can 0006-291X/92 $1.50 1047
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 182, No. 3, 1992 accelerate
resulting chemistry relevance
BIOCHEMICAL
the oxidation Fe(II1) remains of the process discussed.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Fe(I1) to Fe(II1) and that tightly complexed to the casein. is described and the physiological
the The
MATERIALS ARD METHODS a-Casein and dephosphorylated casein were obtained from Sigma Chemical Co. or isolated from fresh skimmed milk (8). All other Iron was assayed by the method of chemicals were reagent grade. Stookey without addition of ascorbate so that only Fe(I1) was Oxidation of Fe(I1) and binding of Fe(II1) product measured (9). to casein was followed by disappearance of Fe(I1) from the reaction A typical reaction mixture contained in a final volume of mixture. loo pg iron as ferrous ammonium sulfate, 1.0 mg casein, 10 ml: 0.05 M piperazine-N,N'-bis[2-ethanesulfonic] (PIPES) buffer, pH 6.5. The solution was maintained at 25OC and gently stirred by a slow stream of air or oxygen (1.2 ml/min) to replenish dissolved oxygen. Aliquots of 0.5 ml were removed and assayed for Fe(I1).
RESULTS Addition of ferrous salts to fresh whole milk results in a rapid oxidation of FeII) to Fe(II1). The oxidation can readily be visualized by the loss of ability of the metal to form colored complexes with Fe(I1) specific chelators or by color formation upon addition of Fe(II1) specific chelators. Xanthine oxidase, an enzyme found in high concentration in milk, can serve as a ferroxidase in animal tissues (lo), and it has been suggested that this enzyme might serve the same function in milk (7). However, oxidation of Fe(I1) by milk is not inhibited by 0.3 mM allopurinol, and neither xanthine oxidase nor lactoperoxidase exhibit any ferrous oxidase activity under my assay conditions (data not shown). Any substance that preferentially binds Fe(II1) to Fe(I1) can serve as a thermodynamic trap for the latter ion and, depending upon the oxidative mechanism, can potentially increase the rate of oxidation (11). The concentrations of casein in human and bovine milk have been reported to be 0.4 and 2.6 g/100 g, respectively 1 shows the acceleration of the oxidation of Fe(I1) (12) - Figure by a-casein at significantly lower concentrations. Dissolved serves as oxidant as demonstrated by the absence of oxygen oxidation when the reaction is carried out anaerobically. Neverthslesss, under my assay conditions the concentration of oxygen is not rate limiting since similar initial rates were 1048
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Minutes
Fioure 1. Effect of casein on iron oxidation. Disappearance of Fe(I1) from the reaction mixture is due to oxidation of the metal to Fe(II1). Reaction conditions are as described in Materials and Methods. Symbols are: (a), no casein; (a), partially dephosphorylated casein, 1.0 mg/ml; (m), casein, 0.5 rag/ml; (A), casein,
1.0 mg/ml;
(O), casein,
2.0 mghul.
observed whether stirring was accomplished by a stream of air or oxygen. Furthermore, Fe(I1) oxidation is dependent upon complexation of the Fe(II1) product by the phosphate groups of evidenced by casein as the observation that partially dephosphorylated casein shows a decreased oxidation rate (Fig. 1). Oxidation of Fe(I1) by casein can not strictly be referred to as a ferroxidase activity because the casein does not act in a catalytic manner. The casein is involved stoichiometrically. Titration of a solution of Fe(I1) with casein shows that approximately 10 pg of iron can be bound per mg of casein (Fig. 2). The oxidized metal is strongly bound to the protein and is not removed by extensive dialysis. The metal can be quantitatively recovered with the protein upon isoelectric precipitation at pH 4.6. Precise kinetic analysis of the casein-mediated oxidation of Fe(I1) is difficult because of the heterogenous nature of the protein and the difficulty in determining the precise number of phosphate groups involved in the reaction. It has been reported that aerobic binding of Fe(I1) to the phosphorylated proteins leads to oxidative destruction of phosphoserine residues accompanied by an increased absorption of ultraviolet light (13). I confirmed The casein mediated oxidation this observation (data not shown). of iron appears to be second order with respect to protein and Fe(I1). Figure 3 shows the pseudo-first-order kinetics of iron 1049
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
67B5 4. 32lOa
0 0.2
014
Milligrams
Ok
016
cash/ml
Ficure 2. Titration of Fe(X) with casein. Reactions conditions are as described in Materials and Methods except that for each increment of added casein the mixture was allowed to staud for 1 hr to ensure that maximum oxidation of Fe(I1) was attained. oxidation in the presence of a high concentration pseudo-first-order rate constant for Fe(I1) calculated by the method of Guggenheim (14) 0.3 min-l, corresponding to a half-life of Fe(I1) minutes.
of casein. The oxidation was and found to be oxidation of 2.4
DISCUSSION In their supplemented
efforts to find a suitable method of producing ironmilk, Hegenauer and co-workers observed that addition
Minutes
Fiaure 3. Initial rate of casein-mediated Fe(I1) oxidation. The reaction conditions are as described in Materials and Methods using casein at a concentration of 2 mg/ml. Insert: pseudofirst-order plot of the rate data by the method of Guggenheim (ref. 14). 1050
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of ferrous sulfate to bovine milk resulted in formation of Fe(III) bound to casein (6). They suggested that Fe(I1) first displaced Ca (II) from the protein with subsequent oxidation of the Fe(II)casein complex. My results support this suggestion. The rate of Fe(I1) oxidation is highly dependent upon the concentration of casein and the rate is pseudo-first-order with respect to Fe(I1) concentration at high concentrations of casein, ruling out a mechanism by which the metal is first oxidized by dissolved oxygen in a rate-limiting step. The observed increase in ultraviolet absorption and the observation that dephosphorylated casein is ineffective in accelerating the oxidation of Fe(I1) demonstrate that the phosphate content of this protein is not only of nutritional importance but may also serve an important function in regulating the oxidation state of iron. The amount of iron in milk varies from species to species with values ranging from 0.1-0.2 mg/l for cow's milk to 5-10 mg/l for rat's milk (15). A large variation among individuals has also been found in human milk, but it is generally agreed that the iron content is approximately 0.5 mg/l. The titration data of Figure 2 demonstrates that the amount of casein in milk of any species can rapidly oxidize and bind all of the iron present and can thus serve as a buffer to ensure that all of the metal is effectively bound to protein. The low concentration of iron in human milk has been a source of consternation to nutritionists fearful of the harmful consequences of iron deficiency in the newborn (16). Currently, the recommended daily allowance of iron for the newborn human is 10 mg which, in view of the actual amount found in human milk, would require iron supplementation for all breast-feeding infants. In contrast, many iron researchers believe that dietary iron is to be avoided in the newborn and serves only to increase the risk of gastro-intestinal tract infection, and that an important function of iron-binding proteins such as lactoferrin is to withhold iron from microorganisms (17). The results presented in this paper may support the latter hypothesis. The high casein concentration of milk ensures rapid oxidation of iron to the trivalent oxidation state with concomitant strong binding to the protein. The net effect make iron less available to intestinal may be to microorganisms as well as to hasten removal of the metal from the intestinal tract by more efficient absorption of the metal-protein complex. 1051
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS This Institute of Health. assistance
work was supported by grant AI-09580 from the National of Allergy and Infectious Diseases, National Institutes The author wishes to acknowledge the expert technical of Ms. Wei Zhang.
REFERENCES 1.
2. 3. 4. 5.
Puschmann, M., and Ganzoni, A.M. (1977) Infect. Immun. 17, 663-664. Alderton, G.W., Ward, V.A., and Fevold, H.L. (1946) Arch. Biochem. 11, 9-13. Arnold, R. R., Cole, M. F., and McGhee, J.R. (1977) Science 197, 263-265. Brock, J.H. (1980) Arch. Dis. Child. 55, 417-421. Griffiths, E., and Humphreys, J. (1977) Infect. Immun. 15, 396-401.
6. Hegenauer, J., Saltman, P., Ludwig, D., Ripley, L., and Ley, A. (1979) J. Agric. Food Chem. 27, 1294-1301. 7. Emery, T. (1987) in Iron Transport in Microbes, Plants and Animals (Winkelmann, G., van der Helm, D., and Neilands, J. B eds.) pp. 235-250, VCH, Weinheim, Federal Republic of G&nany. 8. Hollar, C.M., Law, A.J.R., Dalgleish, D.G., and Brown, R. (1991) J. Dairy Sci. 74, 2403-2409. 9. Stookey, L.L. (1970) Anal. Chem. 42, 779-781. 10. Topham, R-W., Walker, M.C., and Calisch, M.P. (1982) Biochem. Biophys. Res. Commun. 109, 1240-1246. 11. Thorstensen, K., and Aisen, P. (1990) Biochim. Biophys. Acta 1052, 29-35. 12. Jenness, R. (1988) in Fundamentals of Dairy Chemistry, 3d edition (Wong, N.P., ed.), pp. l-37, Van Nostrand Reinhold, NY. 13. Grant, C.T., and Taborsky, G. (1966) Biochemistry 5, 544-555. 14. Hammes, G.G. (1978), Principles of Chemical Kinetics, p. 9, Academic Press, New York. 15. Liinnerdal, B. (1990) in Iron Metabolism in Infants (Lonnerdal, B., Ed.) pp. 87-107, CRC Press, Boca Raton, FL. 16. Scrimshaw, N.S. (1991) Scientific American 265, 46-52. 17 Weinberg, &,. E.D. (1984) Physiol. Rev. 64, 65-102.
1052