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Oxygen lability of cysteine in hemoglobin
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
76,
243-250 (1958)
Oxygen Lability of Cysteine in Hemoglobin C. C. Tsen and A. L. Tappel Prom the Department of Food Technology, University Davis, California
of California,
Received September 9, 1957
INTRODUCTION
The oxidation of proteins by molecular oxygen may be an important deteriorative reaction in vivo and in the storage of biological materials. Hisey and Morrison (1) found that dry hemoglobin took up oxygen in excessof that required to oxidize it to methemoglobin. They suggested that the observed denaturation of globin was caused by its oxidation but provided no direct evidence. Lemberg and Legge (2) and Kikuchi et al. (3) studied the oxidation of hemoglobin in solution, and they suggested that the oxygen uptake in excess of that required for the oxidation to methemoglobin was used in oxidizing some portion of the globin. In their study of the oxidation of myoglobin in solution, George and Stratmann (4) found an oxygen absorption of 2.5 mole oxygen/ mole myoglobin and likewise suggested that oxidation of some oxygenlabile amino acids of the globin must occur. Collectively these workers (14) have provided strong evidence for the oxidation of some portion of the globin of hemoglobin and myoglobin, but they did not determine which of the known oxygen-labile amino acids (5-8) were involved. An important deteriorative mechanism in dehydrated animal tissue is the direct oxidation of protein by molecular oxygen (9). Here also, the labile amino acids were not determined. It is the purpose of this research to broaden our knowledge of the direct oxidation of the protein portion of hemoglobin and to determine which amino acids of the protein are most labile to oxidation. EXPERIMENTAL
Cattle hemoglobin was prepared by a method similar to that of Marri and Singer (10). The hemoglobin solution was freeze-dried, pulverized to a fine powder, and equilibrated to 11 mm. Hg water vapor pressure in nitrogen atmospheres. Pure 243
244
TSEN AND TAPPEL
amino acids (Nutritional Biochemicals Corporation) were dissolved or suspended in ferric chloride solution, freeze-dried, and equilibrated to 11 mm. Hg water vapor pressure. The sulfhydryl reagents, mercuric chloride, iodoacetate, and chloromercuribenzoate were added to the hemoglobin solution before freezedrying or other experimental use. Oxygen absorption was measured manometrically using Warburg flasks wrapped with aluminum foil to exclude light. Sulfhydryl groups of the hemoglobin were measured by the method of Benesch, Lardy, and Benesch (11). The ratio of methemoglobin to total hemoglobin was determined by the procedure of Michel and Harris (12). Total soluble nitrogen of the 0.07 M phosphate buffer, pH 7.0, extract was determined by the Kjeldahl method. Hematin was determined spectrophotometrically as the alkaline pyridine hemochrome. The complete visible spectrum of the 0.07 M phosphate buffer extract was measured, and the loss due to oxidation was determined from absorbance at 500 rnp. RESULTS
Oxidation of Dry Hemoglobin by Molecular Oxygen
Dry hemoglobin oxidized rapidly with an initial rate of 0.26 mole oxygen/mole hemoglobin/hr. and consumed large quantities of oxygen as shown in Table I. During the first 50 hr. the rate of oxygen absorption was relatively constant and then decreased.At the end of 73 days when the experiment was terminated, the oxygen absorption had not reached a maximum value. Similar effects were noted by Hisey and Morrison (1). Identical samples maintained in nitrogen atmospheres showed no gas exchange indicating the absence of physical adsorption. The greatest observed changes shown in Table I were the loss of solubility measured as total soluble nitrogen and spectral absorbance for the hemoglobin reacting with oxygen. This loss of protein solubility TABLE I Changes in Hemoglobin Exposed to Oxygen at 55°C. The hemoglobin used in this experiment differed from the other preparations in that it was not purified by dialysis. The per cent decrease is based on the O-time sample. D;zresegn in Decrease in total Decreasein Time d.SYS
Oxygen consumed mole O*/mole hemoglobin ml./g.
5 75
2.23 6.00
5.6 15.2
soluble nitrogen 02 NX
per cent
36 83
spectral absorbance 02 NZ
per CLW fin cert
8 19
33 61
0,
fier cent
per cent
1 11
36
NZ per cemt
7
OXYGE?r’
LABILITY
OF
215
CYSTEIKE
together with a smaller loss in hematin showed that the globin portion of hemoglobin is most adversely affected by its reaction wit’h oxygen. Direct Oxidation of Cysteine and Histidine
in the Dry State
To ascertain which part of the hemoglobin molecule is most oxygenlabile, hemin and the oxygen-labile amino acids cysteine, tryptophan, tyrosine, methionine, and histidine were exposed to oxygen in the dry state. Dry hemin did not absorb any oxygen. In the dry state without ferric ion catalysis all of these amino acids were stable. In the presence of ferric ion catalysis, cysteine oxidized with an initially fast rate of 0.1 mole oxygen/mole cysteine/hr., which greatly decreased after the first hour giving a total oxygen absorption of 0.2 mole oxygen/mole cysteine in 170 hr. Under the same conditions histidine oxidized with an initial rate of 0.003 mole oxygen/mole histidine/hr. to 0.01 mole oxygen/mole histidine in 170 hr. The other amino acids did not oxidize in the presence of iron. By paper chromatographic separation and comparison with known compounds, the oxidation product of cysteine was identified as cystine. Ko cysteic acid was formed. Because of the small amount of oxidation, the oxidation products of histidine could not be identified. Inhibition
of the Oxidation
of Dry Hemogbok
by Sulfhydryl
Reagents
After cysteine was found to be the most labile amino acid to oxidation in the dry state, it was desirable to determine the oxygen-lability of cysteine in the hemoglobin molecule. Direct measurement showed that our preparation of native hemoglobin contained four sulfhydryl groups per mole of hemoglobin, a result in agreement with that of Ingram (13). After the addition of 4 moles chloromercuribenzoate/mole hemoglobin, no free sulfhydryl groups could be detected. Figure 1 shows that two sulfhydryl reagents gave a maximum of two-thirds inhibition of the oxidation and that this inhibition is an approximate function of the number of sulfhydryl groups bound. Iodoacetate appeared more effective as an inhibitor than chloromercuribenzoate, perhaps because iodoacetate is less specific than chloromercuribenzoate for binding sulfhydryl groups. Titration of the sulfhydryl groups after exposure of the untreated dry hemoglobin to oxygen showed only 8.3 % remaining. Under a nitrogen atmosphere showing no gas exchange there was 75 % remaining, indicating a 25 % loss of sulfhydryl groups caused by the long time exposure.
246
TSEN
AND TAPPEL
0 HOURS FIG. 1. The inhibition of the oxidation of dry hemoglobin by two sulfhydryl reagents. Curve 1 is for gas exchange when dry hemoglobin was held in an atmosphere of pure nitrogen. Curve 7 is the oxygen absorption of 1.5 X 10e6 mole of dry hemoglobin in pure oxygen at 37°C. Curves 3,5, and 6 are for oxygen absorption of dry hemoglobin with chloromercuribeneoate concentrations of 4, 2, and 1 moles chloromercuribenzoate/mole hemoglobin. Curves 2 and 4 are for oxygen absorption of dry hemoglobin with iodoacetate concentrations of 4 and 2 moles iodoacetate/mole hemoglobin.
E$ect of pH, Water and Temperature on the Oxidation of Dry Hemoglobin During
the freeze-drying
methemoglobin
of hemoglobin,
before oxidation
it was 90 % oxidized
in the manometric
to
vessels commenced.
After exposure of hemoglobin, whose aqueous pH is shown in Table II, to oxygen for 134 hr., there was no change in the amount of methemoglobin indicating that all of the oxygen absorbed oxidized sulfhydryl and other labile groups of the protein. The results in Table II show that the rate of oxidation and the loss of sulfhydryl groups was increased by increased pH. This effect may be ascribed to the pK, of 10.3 of the sulfhydryl group where it is most oxygen-labile. The rate of oxygen absorption was not greatly affected by the water
OXYGEN
LABILITY
OF
247
CYSTEINE
TABLE II Effect of the Aqueous pH on the Om’dation of Dry Hemoglobin at WC. The pH values given in this table were those of the hemoglobin solution obtained by adjusting with 0.6 M phosphate buffers before the freeze-drying. The initial rate was constant for 50 hr. The total oxidation period was 134 hr. PB
Initial rate mole O*/mole hemoglobin/hr.
9.60 8.07 6.82 5.95
0.024 0.019 0.012 0.007
Total oxygen absorbed mole 02/male
Residual sulfhydryl groups
hemoglobin
2.28 1.92 1.42 1.19
per cent
12.6 17.8 36.4 40.1
content of the dry hemoglobin. The rates for the first 50 hr. were 0.010, 0.013, 0.014, 0.009 mole oxygen/mole hemoglobin/hr. for water contents of 3.5, 4.2, 9.6, 17.5 % water, respectively. This oxidation was measured at pH 6.9 and 37°C. The rate of oxygen absorption increased as a function of temperature. The rate for the first 50 hr. was 0.006, 0.015, 0.028 mole oxygen/mole hemoglobin/hr. at temperatures of 15, 37, and 45”C., respectively. From the Arrhenius relationship the activation energy was found to be 9.6 kcal./mole. Oxidation of Hemoglobin in Solution As is well known, oxyhemoglobin rapidly deoxygenated at low pH and was oxidized to methemoglobin. In contrast, at high pH there was no oxygen evolution but actually oxygen absorption with little formation of methemoglobin. The most important results shown in Table III are the direct measurements of the oxidation of the sulfhydryl groups of hemoglobin, which increases with increasing pH. The sulfhydryl reagents mercuric chloride and chloromercuribenzoate were added to 2.5 X 1O-6 M oxyhemoglobinin 0.6 M phosphate buffer of pH 6.5, and this system was allowed to deoxygenate at 37°C. After 3 hr. the control sample showed 1.1 mole oxygen evolved/mole oxyhemoglobin. With 2 moles mercuric chloride or 4 moles chloromercuribenzoate/mole oxyhemoglobin, there were 1.5 moles oxygen evolved. When the amount of inhibitor was increased to 3 moles mercuric chloride and G moles chloromercuribenzoate, the oxygen evolution was 1.6 moles oxygen/mole oxyhemoglobin. These results show that a large amount of the oxygen absorbed in the oxidation of the protein is reacting with
248
TSEN AND TAPPEL
TABLE III Efect of pH on the Oxidation of Hemoglobin in Solution at WC. The oxyhemoglobin concentration was 2.56 X 1O-6M in 0.6 M phosphate buffer. The oxyhemoglobin contained 3% methemoglobin at the initiation of oxidation. PH
Initial
rate
of oxygen
mole Oe/mole min.
4.85 5.13 5.48 5.90 6.50 7.00 7.55 8.20 8.90
evolution
Methemoglobin
oxyhemoglobin/ X 108
38 30 21 14 9.4 5.0 -1.4 -2.6 -5.0
Changes due to oxidation formed Sulfhydryl
group
per cent
per cent
93 91 89 85 68 48 38 28 26
0.9 1.3 1.3 1.8 2.1 2.4 3.0 3.9 4.7
loss
the sulfhydryl groups which may be specifically blocked by the stoichiometric amount of sulfhydryl reagents. DISCUSSION
The results of this study showing oxidation of the protein portion of hemoglobin are in general agreement with those of Hisey and Morrison (l), Lemberg and Legge (2), George and Stratmann (4), and Kikuchi et al. (3), except that these previous investigators did not determine which groups of the protein were labile to oxidation. This study confirms and extends the work of others showing that dry hemoglobin is very labile to oxidation. It is converted to methemoglobin during freezedrying, and thereafter on exposure to oxygen absorbs large amounts of oxygen. The significant loss of solubility of the oxidized hemoglobin definitely indicates denaturation of the protein. There was no evidence that the oxidation of the iron protoporphyrin of hemoglobin can account for the large amounts of oxygen absorbed, and, further, dry hemin was stable in the presence of oxygen. The oxidation of cysteine in solution in the presence of metal catalysis is well known (14), and this research shows that this oxygen-lability holds for dry cysteine in the presence of ferric iron. The most significant feature of this research is the demonstration that the sulfhydryl group of hemoglobin is the most labile group and the one responsible for the largest portion of the oxygen absorbed. Although it is well known that the sulfhydryl groups of hemo-
OXYGEN
LABILITY
OF CYSTEINE
249
globin can be oxidized by a number of chemical reagents, such as ferricyanide, this is the first report of the direct oxidation by molecular oxygen. The first step in the mechanism of this oxidation may be visualized as an oxidation of the sulfhydryl pairs, which form neighboring groups in the native hemoglobin (14), to form disulfide bonds. However, the large amounts of oxygen absorption observed in this study to result when the sulfhydryl groups are free cannot be accounted for on the basis of sulfhydryl oxidation to disulfide, and this oxidation must proceed further. Other oxygen-labile amino acids such as histidine, tryptophan, tyrosine, and methionine are probably responsible for the small amount of oxygen which cannot be accounted for as cysteine oxidation. SUMMARY
This study extends and confirms the work of others showing that hemoglobin is very labile to oxidation. The significant loss of solubility of the oxidized dry hemoglobin indicates denaturation of the protein. To ascertain which part of the hemoglobin molecule is responsible for the oxidation, hemin and the oxygen-labile amino acids in the presence of ferric chloride have been exposed to oxygen in the dry state. Among them, cysteine and histidine have been shown to be oxygen-labile. The oxidation product of cysteine is cystine. There was a significant loss of sulfhydryl groups in hemoglobin after oxidation. Also, the largest portion of the oxygen uptake can be inhibited by blocking the sulfhydryl groups. This inhibition is an approximate function of the number of sulfhydryl groups blocked. The sulfhydryl group of hemoglobin is the most labile group to oxygen in the dry state and in solution. In the dry stat’e, the rate of the oxidation is increased with increasing temperature and with increasing pH of the hemoglobin solutions before the freeze-drying. REFERENCES 1. HISEY, A., AND MORRISON, 2. LEMBERC:, R., AND LEGGE, 3. 4. 5.
6. 7.
I). B., J. Biol. Chem. 130, 763 (1939). J. W., “Hematin Compounds 2nd Bile Pigments.” Intcrscience, New York, 1949. KIKUCHI, G., SHKJKCYA, It., SUZECKI, RI., AND NAKA>\II:RA, C., J. ~iochert/. (Tokyo) 42, 267 (1955). GEORGE, P., AND STRATMANS, C. J., Biochem. J. 61, 103, (1952). BENNETT, M. A., AND TOENNIES, G., J. Bid. Chem. 146, 671 (1942). TOENNIES, G., J. Biol. Chem. 146, 667 (1942). WEIL. T,., AND BUCHERT, A. It., Such. Biochen~. Riophys. 34, 11 (1951).
250
TSEN
AND
TAPPEL
8. WEIL, L., GORDON, W. G., AND BUCHERT, A. R., Arch. Biochem.
9. 10. 11. 12. 13. 14.
Biophys.
88,
90 (1951). TAPPEL, A. L., Food Research 21, 195 (1956). MARRI, M. S., AND SINGER, K. Arch. Biochem. Biophys. 68, 414 (1955). BENESCH, R. E., LARDY, H. A., AND BENESCH, R., J. Biol. Chem. 216, 663 (1955). MICHEL, H. O., AND HARRIS, J. S., J. Lab. Clin. Med. 26, 445 (1940). INGRAM, V. M., Biochem. J. 69, 653 (1955). WARBURG, O., AND SAKUMA, S., Arch. ges. Physiol., Pfltigers 200, 203 (1923).
OF BIOCHEMISTRY
AND
BIOPHYSICS
76,
243-250 (1958)
Oxygen Lability of Cysteine in Hemoglobin C. C. Tsen and A. L. Tappel Prom the Department of Food Technology, University Davis, California
of California,
Received September 9, 1957
INTRODUCTION
The oxidation of proteins by molecular oxygen may be an important deteriorative reaction in vivo and in the storage of biological materials. Hisey and Morrison (1) found that dry hemoglobin took up oxygen in excessof that required to oxidize it to methemoglobin. They suggested that the observed denaturation of globin was caused by its oxidation but provided no direct evidence. Lemberg and Legge (2) and Kikuchi et al. (3) studied the oxidation of hemoglobin in solution, and they suggested that the oxygen uptake in excess of that required for the oxidation to methemoglobin was used in oxidizing some portion of the globin. In their study of the oxidation of myoglobin in solution, George and Stratmann (4) found an oxygen absorption of 2.5 mole oxygen/ mole myoglobin and likewise suggested that oxidation of some oxygenlabile amino acids of the globin must occur. Collectively these workers (14) have provided strong evidence for the oxidation of some portion of the globin of hemoglobin and myoglobin, but they did not determine which of the known oxygen-labile amino acids (5-8) were involved. An important deteriorative mechanism in dehydrated animal tissue is the direct oxidation of protein by molecular oxygen (9). Here also, the labile amino acids were not determined. It is the purpose of this research to broaden our knowledge of the direct oxidation of the protein portion of hemoglobin and to determine which amino acids of the protein are most labile to oxidation. EXPERIMENTAL
Cattle hemoglobin was prepared by a method similar to that of Marri and Singer (10). The hemoglobin solution was freeze-dried, pulverized to a fine powder, and equilibrated to 11 mm. Hg water vapor pressure in nitrogen atmospheres. Pure 243
244
TSEN AND TAPPEL
amino acids (Nutritional Biochemicals Corporation) were dissolved or suspended in ferric chloride solution, freeze-dried, and equilibrated to 11 mm. Hg water vapor pressure. The sulfhydryl reagents, mercuric chloride, iodoacetate, and chloromercuribenzoate were added to the hemoglobin solution before freezedrying or other experimental use. Oxygen absorption was measured manometrically using Warburg flasks wrapped with aluminum foil to exclude light. Sulfhydryl groups of the hemoglobin were measured by the method of Benesch, Lardy, and Benesch (11). The ratio of methemoglobin to total hemoglobin was determined by the procedure of Michel and Harris (12). Total soluble nitrogen of the 0.07 M phosphate buffer, pH 7.0, extract was determined by the Kjeldahl method. Hematin was determined spectrophotometrically as the alkaline pyridine hemochrome. The complete visible spectrum of the 0.07 M phosphate buffer extract was measured, and the loss due to oxidation was determined from absorbance at 500 rnp. RESULTS
Oxidation of Dry Hemoglobin by Molecular Oxygen
Dry hemoglobin oxidized rapidly with an initial rate of 0.26 mole oxygen/mole hemoglobin/hr. and consumed large quantities of oxygen as shown in Table I. During the first 50 hr. the rate of oxygen absorption was relatively constant and then decreased.At the end of 73 days when the experiment was terminated, the oxygen absorption had not reached a maximum value. Similar effects were noted by Hisey and Morrison (1). Identical samples maintained in nitrogen atmospheres showed no gas exchange indicating the absence of physical adsorption. The greatest observed changes shown in Table I were the loss of solubility measured as total soluble nitrogen and spectral absorbance for the hemoglobin reacting with oxygen. This loss of protein solubility TABLE I Changes in Hemoglobin Exposed to Oxygen at 55°C. The hemoglobin used in this experiment differed from the other preparations in that it was not purified by dialysis. The per cent decrease is based on the O-time sample. D;zresegn in Decrease in total Decreasein Time d.SYS
Oxygen consumed mole O*/mole hemoglobin ml./g.
5 75
2.23 6.00
5.6 15.2
soluble nitrogen 02 NX
per cent
36 83
spectral absorbance 02 NZ
per CLW fin cert
8 19
33 61
0,
fier cent
per cent
1 11
36
NZ per cemt
7
OXYGE?r’
LABILITY
OF
215
CYSTEIKE
together with a smaller loss in hematin showed that the globin portion of hemoglobin is most adversely affected by its reaction wit’h oxygen. Direct Oxidation of Cysteine and Histidine
in the Dry State
To ascertain which part of the hemoglobin molecule is most oxygenlabile, hemin and the oxygen-labile amino acids cysteine, tryptophan, tyrosine, methionine, and histidine were exposed to oxygen in the dry state. Dry hemin did not absorb any oxygen. In the dry state without ferric ion catalysis all of these amino acids were stable. In the presence of ferric ion catalysis, cysteine oxidized with an initially fast rate of 0.1 mole oxygen/mole cysteine/hr., which greatly decreased after the first hour giving a total oxygen absorption of 0.2 mole oxygen/mole cysteine in 170 hr. Under the same conditions histidine oxidized with an initial rate of 0.003 mole oxygen/mole histidine/hr. to 0.01 mole oxygen/mole histidine in 170 hr. The other amino acids did not oxidize in the presence of iron. By paper chromatographic separation and comparison with known compounds, the oxidation product of cysteine was identified as cystine. Ko cysteic acid was formed. Because of the small amount of oxidation, the oxidation products of histidine could not be identified. Inhibition
of the Oxidation
of Dry Hemogbok
by Sulfhydryl
Reagents
After cysteine was found to be the most labile amino acid to oxidation in the dry state, it was desirable to determine the oxygen-lability of cysteine in the hemoglobin molecule. Direct measurement showed that our preparation of native hemoglobin contained four sulfhydryl groups per mole of hemoglobin, a result in agreement with that of Ingram (13). After the addition of 4 moles chloromercuribenzoate/mole hemoglobin, no free sulfhydryl groups could be detected. Figure 1 shows that two sulfhydryl reagents gave a maximum of two-thirds inhibition of the oxidation and that this inhibition is an approximate function of the number of sulfhydryl groups bound. Iodoacetate appeared more effective as an inhibitor than chloromercuribenzoate, perhaps because iodoacetate is less specific than chloromercuribenzoate for binding sulfhydryl groups. Titration of the sulfhydryl groups after exposure of the untreated dry hemoglobin to oxygen showed only 8.3 % remaining. Under a nitrogen atmosphere showing no gas exchange there was 75 % remaining, indicating a 25 % loss of sulfhydryl groups caused by the long time exposure.
246
TSEN
AND TAPPEL
0 HOURS FIG. 1. The inhibition of the oxidation of dry hemoglobin by two sulfhydryl reagents. Curve 1 is for gas exchange when dry hemoglobin was held in an atmosphere of pure nitrogen. Curve 7 is the oxygen absorption of 1.5 X 10e6 mole of dry hemoglobin in pure oxygen at 37°C. Curves 3,5, and 6 are for oxygen absorption of dry hemoglobin with chloromercuribeneoate concentrations of 4, 2, and 1 moles chloromercuribenzoate/mole hemoglobin. Curves 2 and 4 are for oxygen absorption of dry hemoglobin with iodoacetate concentrations of 4 and 2 moles iodoacetate/mole hemoglobin.
E$ect of pH, Water and Temperature on the Oxidation of Dry Hemoglobin During
the freeze-drying
methemoglobin
of hemoglobin,
before oxidation
it was 90 % oxidized
in the manometric
to
vessels commenced.
After exposure of hemoglobin, whose aqueous pH is shown in Table II, to oxygen for 134 hr., there was no change in the amount of methemoglobin indicating that all of the oxygen absorbed oxidized sulfhydryl and other labile groups of the protein. The results in Table II show that the rate of oxidation and the loss of sulfhydryl groups was increased by increased pH. This effect may be ascribed to the pK, of 10.3 of the sulfhydryl group where it is most oxygen-labile. The rate of oxygen absorption was not greatly affected by the water
OXYGEN
LABILITY
OF
247
CYSTEINE
TABLE II Effect of the Aqueous pH on the Om’dation of Dry Hemoglobin at WC. The pH values given in this table were those of the hemoglobin solution obtained by adjusting with 0.6 M phosphate buffers before the freeze-drying. The initial rate was constant for 50 hr. The total oxidation period was 134 hr. PB
Initial rate mole O*/mole hemoglobin/hr.
9.60 8.07 6.82 5.95
0.024 0.019 0.012 0.007
Total oxygen absorbed mole 02/male
Residual sulfhydryl groups
hemoglobin
2.28 1.92 1.42 1.19
per cent
12.6 17.8 36.4 40.1
content of the dry hemoglobin. The rates for the first 50 hr. were 0.010, 0.013, 0.014, 0.009 mole oxygen/mole hemoglobin/hr. for water contents of 3.5, 4.2, 9.6, 17.5 % water, respectively. This oxidation was measured at pH 6.9 and 37°C. The rate of oxygen absorption increased as a function of temperature. The rate for the first 50 hr. was 0.006, 0.015, 0.028 mole oxygen/mole hemoglobin/hr. at temperatures of 15, 37, and 45”C., respectively. From the Arrhenius relationship the activation energy was found to be 9.6 kcal./mole. Oxidation of Hemoglobin in Solution As is well known, oxyhemoglobin rapidly deoxygenated at low pH and was oxidized to methemoglobin. In contrast, at high pH there was no oxygen evolution but actually oxygen absorption with little formation of methemoglobin. The most important results shown in Table III are the direct measurements of the oxidation of the sulfhydryl groups of hemoglobin, which increases with increasing pH. The sulfhydryl reagents mercuric chloride and chloromercuribenzoate were added to 2.5 X 1O-6 M oxyhemoglobinin 0.6 M phosphate buffer of pH 6.5, and this system was allowed to deoxygenate at 37°C. After 3 hr. the control sample showed 1.1 mole oxygen evolved/mole oxyhemoglobin. With 2 moles mercuric chloride or 4 moles chloromercuribenzoate/mole oxyhemoglobin, there were 1.5 moles oxygen evolved. When the amount of inhibitor was increased to 3 moles mercuric chloride and G moles chloromercuribenzoate, the oxygen evolution was 1.6 moles oxygen/mole oxyhemoglobin. These results show that a large amount of the oxygen absorbed in the oxidation of the protein is reacting with
248
TSEN AND TAPPEL
TABLE III Efect of pH on the Oxidation of Hemoglobin in Solution at WC. The oxyhemoglobin concentration was 2.56 X 1O-6M in 0.6 M phosphate buffer. The oxyhemoglobin contained 3% methemoglobin at the initiation of oxidation. PH
Initial
rate
of oxygen
mole Oe/mole min.
4.85 5.13 5.48 5.90 6.50 7.00 7.55 8.20 8.90
evolution
Methemoglobin
oxyhemoglobin/ X 108
38 30 21 14 9.4 5.0 -1.4 -2.6 -5.0
Changes due to oxidation formed Sulfhydryl
group
per cent
per cent
93 91 89 85 68 48 38 28 26
0.9 1.3 1.3 1.8 2.1 2.4 3.0 3.9 4.7
loss
the sulfhydryl groups which may be specifically blocked by the stoichiometric amount of sulfhydryl reagents. DISCUSSION
The results of this study showing oxidation of the protein portion of hemoglobin are in general agreement with those of Hisey and Morrison (l), Lemberg and Legge (2), George and Stratmann (4), and Kikuchi et al. (3), except that these previous investigators did not determine which groups of the protein were labile to oxidation. This study confirms and extends the work of others showing that dry hemoglobin is very labile to oxidation. It is converted to methemoglobin during freezedrying, and thereafter on exposure to oxygen absorbs large amounts of oxygen. The significant loss of solubility of the oxidized hemoglobin definitely indicates denaturation of the protein. There was no evidence that the oxidation of the iron protoporphyrin of hemoglobin can account for the large amounts of oxygen absorbed, and, further, dry hemin was stable in the presence of oxygen. The oxidation of cysteine in solution in the presence of metal catalysis is well known (14), and this research shows that this oxygen-lability holds for dry cysteine in the presence of ferric iron. The most significant feature of this research is the demonstration that the sulfhydryl group of hemoglobin is the most labile group and the one responsible for the largest portion of the oxygen absorbed. Although it is well known that the sulfhydryl groups of hemo-
OXYGEN
LABILITY
OF CYSTEINE
249
globin can be oxidized by a number of chemical reagents, such as ferricyanide, this is the first report of the direct oxidation by molecular oxygen. The first step in the mechanism of this oxidation may be visualized as an oxidation of the sulfhydryl pairs, which form neighboring groups in the native hemoglobin (14), to form disulfide bonds. However, the large amounts of oxygen absorption observed in this study to result when the sulfhydryl groups are free cannot be accounted for on the basis of sulfhydryl oxidation to disulfide, and this oxidation must proceed further. Other oxygen-labile amino acids such as histidine, tryptophan, tyrosine, and methionine are probably responsible for the small amount of oxygen which cannot be accounted for as cysteine oxidation. SUMMARY
This study extends and confirms the work of others showing that hemoglobin is very labile to oxidation. The significant loss of solubility of the oxidized dry hemoglobin indicates denaturation of the protein. To ascertain which part of the hemoglobin molecule is responsible for the oxidation, hemin and the oxygen-labile amino acids in the presence of ferric chloride have been exposed to oxygen in the dry state. Among them, cysteine and histidine have been shown to be oxygen-labile. The oxidation product of cysteine is cystine. There was a significant loss of sulfhydryl groups in hemoglobin after oxidation. Also, the largest portion of the oxygen uptake can be inhibited by blocking the sulfhydryl groups. This inhibition is an approximate function of the number of sulfhydryl groups blocked. The sulfhydryl group of hemoglobin is the most labile group to oxygen in the dry state and in solution. In the dry stat’e, the rate of the oxidation is increased with increasing temperature and with increasing pH of the hemoglobin solutions before the freeze-drying. REFERENCES 1. HISEY, A., AND MORRISON, 2. LEMBERC:, R., AND LEGGE, 3. 4. 5.
6. 7.
I). B., J. Biol. Chem. 130, 763 (1939). J. W., “Hematin Compounds 2nd Bile Pigments.” Intcrscience, New York, 1949. KIKUCHI, G., SHKJKCYA, It., SUZECKI, RI., AND NAKA>\II:RA, C., J. ~iochert/. (Tokyo) 42, 267 (1955). GEORGE, P., AND STRATMANS, C. J., Biochem. J. 61, 103, (1952). BENNETT, M. A., AND TOENNIES, G., J. Bid. Chem. 146, 671 (1942). TOENNIES, G., J. Biol. Chem. 146, 667 (1942). WEIL. T,., AND BUCHERT, A. It., Such. Biochen~. Riophys. 34, 11 (1951).
250
TSEN
AND
TAPPEL
8. WEIL, L., GORDON, W. G., AND BUCHERT, A. R., Arch. Biochem.
9. 10. 11. 12. 13. 14.
Biophys.
88,
90 (1951). TAPPEL, A. L., Food Research 21, 195 (1956). MARRI, M. S., AND SINGER, K. Arch. Biochem. Biophys. 68, 414 (1955). BENESCH, R. E., LARDY, H. A., AND BENESCH, R., J. Biol. Chem. 216, 663 (1955). MICHEL, H. O., AND HARRIS, J. S., J. Lab. Clin. Med. 26, 445 (1940). INGRAM, V. M., Biochem. J. 69, 653 (1955). WARBURG, O., AND SAKUMA, S., Arch. ges. Physiol., Pfltigers 200, 203 (1923).