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The use of ferricyanide for the preparation of methaemoglobin
284
BIOCHIMICA ET BIOPHYSICAACTA
BBA Report BBA 31094
The use of ferricyanide for the preparation of methaemoglobin DENNIS GEYER and R. LEMBERG The Institute of Medical Research, The Royal North Shore Hospital of Sydney, Crows Nest, N.S.W. 2065 (,4ustralia)
(Received October 30th, 1970)
SUMMARY A warning is issued concerning the use of potassium ferricyanide in the preparation of methaemoglobin. Standard dialysis procedures are insufficient to remove ferrocyanide ions and an improved method of purification of methaemoglobin is suggested.
Potassium ferricyanide is widely used to prepare methaemoglobin. It is common to remove excess ferricyanide from such preparations by dialysis (e.g. ref. 1) but we wish to emphasize that in this type of procedure ferrocyanide remains bound to the protein. We encountered this phenomenon when we tried to remove cyanide from preparations of purified cytochrome oxidase 2 by dialysis against methaemoglobin prepared in the presence of ferricyanide and extensively dialysed against 0.1 M phosphate buffer (pH 7.4). However, we found that the methaemoglobin supplied cyanide to hitherto cyanide-free oxidase preparations. Cyanide contents, before and after dialysis with methaemoglobin, were measured quantitatively after acid hydrolysis of cytochrome oxidase a . Ferrocyanide, which is a strong combinant and precipitant of basic proteins including globin4 , is produced in the oxidation of oxyhaemoglobin by potassium ferricyanide. Since the dissociation constant for cyanide from ferrocyanide ion is greater than from ferricyanide ion s , it was suspected that residual ferrocyanide, which had not been removed by dialysis, was providing cyanide ions. Methaemoglobjn can be prepared and separated from ferrocyanide in the following way. 10 ml oxyhaemoglobin ( 5 . 1 0 -3 M) is incubated with a 3 M excess of potassium ferricyanide at 20 ° for 10 min. The resultant solution is passed through a Sephadex G-25 (fine) column (Pharmacia, Uppsala; dimensions: 26 cm × 4 cm), equilibrated with 0.1 M phosphate buffer (pH 7.4). The first three quarters of the brown methaemoglobin fraction is collected. By using colorimetric tests for ferrocyanide (ref. 6; both FeC13 and AgNO3) we found, with the above column, that methaemoglobin (brown), ferrocyanide (colourless) and ferricyanide (yellow) formed three successive bands with some overlapping. The Biochim. Biophvs. Acta, 229 (1971) 284-285
BBA REPORT
285
methaemoglobin, purified in this way, no longer contaminated previously untreated oxidase preparations with cyanide and was itself free of cyanide. There has been a considerable amount of confusion concerning the products of various methods of preparing methaemoglobin 7. Spectral differences have been observed between methaemoglobin prepared using potassium ferricyanide and methaemoglobin prepared using sodium nitrite but these differences have been explained in terms of the formation of a nitrite complex 8'9. On the other hand, ferrocyanide, which is produced in the preparation, has been shown to alter the kinetics of reduction by ascorbic acid of methaemoglobin 1° . Ferrocyanide may similarly be involved in altering the kinetics of oxidation of reduced cytochrome c by ferricyanide 11 . The fact that Remmer 12 has been able to isolate a complex between ferrocyanide and methaemoglobin, which cannot be dissociated by standard dialysis procedures substantiates these observations and is consistent with our findings. The use of potassium ferricyanide in the preparation of methaemoglobin is thus seen to introduce difficulties. We emphasize that either adequate precautions must be taken to remove ferrocyanide as well as excess ferricyanide, or that methaemoglobin is prepared by autoxidation of haemoglobin at low pH 7 . Unfortunately, this method is not ideal since the process is slow and may possibly lead to some alteration of the molecule. REFERENCES 1 B.F. Cameron and P. George, Biochim. Biophys. Acta, 194 (1969) 16. 2 G.E. Mansley, J.T. Stanbury and R. Lemberg, Biochim. Biophys. Acta, 113 (1966) 33. 3 J. Epstein, Anal, Chem., 19 (1947) 272. 4 C. Ivar Bang, Z. Physiol. Chem., 27 (1899) 463. 5 K.B. Yatsimirskii and V.P. Vasil'ev, Instability Constants of Complex Compounds, Pergamon Press, 1960, p.108. 6 A.I. Vogel, Macro and Semimicro Qualitative Inorganic Analysis, Longmans, London, 1960, p.348. 7 R. Lemberg and J.W. Legge, Hematin Compounds and Bile Pigments, Interscience, New York, 1949, p.389. 8 D.W. Van Assendelft and W.G. Zijlstra, Clin. Chim. Acta, 11 (1965) 571. 9 G.B. Theil and J.E. Auer, Clin. Chem., 13 (1967) 1010. 10 Q.H. Gibson, Biochem. J., 37 (1943) 615. 11 B. Chance, Federation Proc., 29 (1970) Abstr. 2762. 12 H. Remmer, Biochem. Z., 330 (1958) 232. Biochim, Biophy~ Aeta, 229 (1971) 284-285
BIOCHIMICA ET BIOPHYSICAACTA
BBA Report BBA 31094
The use of ferricyanide for the preparation of methaemoglobin DENNIS GEYER and R. LEMBERG The Institute of Medical Research, The Royal North Shore Hospital of Sydney, Crows Nest, N.S.W. 2065 (,4ustralia)
(Received October 30th, 1970)
SUMMARY A warning is issued concerning the use of potassium ferricyanide in the preparation of methaemoglobin. Standard dialysis procedures are insufficient to remove ferrocyanide ions and an improved method of purification of methaemoglobin is suggested.
Potassium ferricyanide is widely used to prepare methaemoglobin. It is common to remove excess ferricyanide from such preparations by dialysis (e.g. ref. 1) but we wish to emphasize that in this type of procedure ferrocyanide remains bound to the protein. We encountered this phenomenon when we tried to remove cyanide from preparations of purified cytochrome oxidase 2 by dialysis against methaemoglobin prepared in the presence of ferricyanide and extensively dialysed against 0.1 M phosphate buffer (pH 7.4). However, we found that the methaemoglobin supplied cyanide to hitherto cyanide-free oxidase preparations. Cyanide contents, before and after dialysis with methaemoglobin, were measured quantitatively after acid hydrolysis of cytochrome oxidase a . Ferrocyanide, which is a strong combinant and precipitant of basic proteins including globin4 , is produced in the oxidation of oxyhaemoglobin by potassium ferricyanide. Since the dissociation constant for cyanide from ferrocyanide ion is greater than from ferricyanide ion s , it was suspected that residual ferrocyanide, which had not been removed by dialysis, was providing cyanide ions. Methaemoglobjn can be prepared and separated from ferrocyanide in the following way. 10 ml oxyhaemoglobin ( 5 . 1 0 -3 M) is incubated with a 3 M excess of potassium ferricyanide at 20 ° for 10 min. The resultant solution is passed through a Sephadex G-25 (fine) column (Pharmacia, Uppsala; dimensions: 26 cm × 4 cm), equilibrated with 0.1 M phosphate buffer (pH 7.4). The first three quarters of the brown methaemoglobin fraction is collected. By using colorimetric tests for ferrocyanide (ref. 6; both FeC13 and AgNO3) we found, with the above column, that methaemoglobin (brown), ferrocyanide (colourless) and ferricyanide (yellow) formed three successive bands with some overlapping. The Biochim. Biophvs. Acta, 229 (1971) 284-285
BBA REPORT
285
methaemoglobin, purified in this way, no longer contaminated previously untreated oxidase preparations with cyanide and was itself free of cyanide. There has been a considerable amount of confusion concerning the products of various methods of preparing methaemoglobin 7. Spectral differences have been observed between methaemoglobin prepared using potassium ferricyanide and methaemoglobin prepared using sodium nitrite but these differences have been explained in terms of the formation of a nitrite complex 8'9. On the other hand, ferrocyanide, which is produced in the preparation, has been shown to alter the kinetics of reduction by ascorbic acid of methaemoglobin 1° . Ferrocyanide may similarly be involved in altering the kinetics of oxidation of reduced cytochrome c by ferricyanide 11 . The fact that Remmer 12 has been able to isolate a complex between ferrocyanide and methaemoglobin, which cannot be dissociated by standard dialysis procedures substantiates these observations and is consistent with our findings. The use of potassium ferricyanide in the preparation of methaemoglobin is thus seen to introduce difficulties. We emphasize that either adequate precautions must be taken to remove ferrocyanide as well as excess ferricyanide, or that methaemoglobin is prepared by autoxidation of haemoglobin at low pH 7 . Unfortunately, this method is not ideal since the process is slow and may possibly lead to some alteration of the molecule. REFERENCES 1 B.F. Cameron and P. George, Biochim. Biophys. Acta, 194 (1969) 16. 2 G.E. Mansley, J.T. Stanbury and R. Lemberg, Biochim. Biophys. Acta, 113 (1966) 33. 3 J. Epstein, Anal, Chem., 19 (1947) 272. 4 C. Ivar Bang, Z. Physiol. Chem., 27 (1899) 463. 5 K.B. Yatsimirskii and V.P. Vasil'ev, Instability Constants of Complex Compounds, Pergamon Press, 1960, p.108. 6 A.I. Vogel, Macro and Semimicro Qualitative Inorganic Analysis, Longmans, London, 1960, p.348. 7 R. Lemberg and J.W. Legge, Hematin Compounds and Bile Pigments, Interscience, New York, 1949, p.389. 8 D.W. Van Assendelft and W.G. Zijlstra, Clin. Chim. Acta, 11 (1965) 571. 9 G.B. Theil and J.E. Auer, Clin. Chem., 13 (1967) 1010. 10 Q.H. Gibson, Biochem. J., 37 (1943) 615. 11 B. Chance, Federation Proc., 29 (1970) Abstr. 2762. 12 H. Remmer, Biochem. Z., 330 (1958) 232. Biochim, Biophy~ Aeta, 229 (1971) 284-285