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Sedimentation behavior of horse carbon monoxide hemoglobin at low pH in the presence of mercaptoethanol and urea
ARCHIVES
OF
BIOCHEMIBTRY
AND
BIOPHYSICS
71, 342-345 (1957)
Sedimentation Behavior of Horse Carbon Monoxide Hemoglobin at Low pH in the Presence of Mercaptoethanol and Urea Frederick
J. Gutter,
Herbert
A. Sober and Elbert A. Peterson
From the Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Public Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland Received January
11, 1957
In a previous communication (1) the effect of mercaptoethanol and urea upon the dissociation of hemoglobin between pH 5.8 and 6.6 was reported. The present study compares the effect of pH, at values ranging from 4.0 to 7.2, on the sedimentation of horse hemoglobin in the presence of mercaptoethanol, urea, and their combination, with that observed over a similar pH range in potassium chloride solution alone. METHODS
AND MATERIALS
Horse carbon monoxide hemoglobin (COHb) was prepared by ammonium sulfate fractionation, as in Ref. (l), except for precipitation by 90% saturated rather than 52% saturated ammonium sulfate. The preparation was lyophilized and stored in the cold in an atmosphere of carbon monoxide, and was essentially homogeneous in the analytical ultracentrifuge as well as in moving-boundary electrophoresis at pH 8.6. Baker’s Analyzed Reagent KCl, 2-mercaptoethanol (Eastman Organic Chemicals), and Merckurea (reagent grade, giving a negative Nessler’s test) were employed. Hemoglobin solutions were prepared at a concentration of 0.7% (W/V) in 0.1 M KCI, 0.1 M KC14 M urea, 0.1 M KCl-1.4 M mercaptoethanol, or 0.1 M KC14 1M urea-l.4 M mercaptoethanol. As demonstrated by Steinhardt (a), and corroborated in this study, no further reduction in sedimentation constant was obtained by increasing the urea concentration from 4.0 to 7.5 M at neutral PH. After adjustment with 0.05 N HCl to the appropriate pH, the solutions were dialyzed for 24 hr. at 5°C. on a rocking dialyzer against the same solvent, similarly adjusted. The Spinco synthetic boundary cell (3), and model E analytical ultracentrifuge were used for all measurements. Rotor speed was 59,780 r.p.m. Photographic con342
SEDIMENTATION
OF CARBON MONOXIDE
HEMOGLOBIN
343
ditions, the method of measuring sedimentation constants (SN,~), and the corrections for viscosity, density, temperature, and solution in water have been described (1). The question of the validity of the correction of sedimentation coefficients for viscosity and density in multicomponent systems, according to the Svedberg equation, was considered in the same paper. Correction was also made for the adiabatic cooling of the ultracentrifuge rotor (4, 5). All ultracentrifuge runs were made at room temperature. RESULTS
AND DISCUSSION
The data obtained are shown in Fig. 1. There is good agreement between our results for horse COHb in KC1 alone and those of Field and O’Brien (6) for dissociation of human hemoglobin at low pH. At the lowest pH studied, 3.5, an S20,Wof 2.9 was obtained in this solvent. When the pH was subsequently raised to 7.5, only about 40% of the hemoglobin remained in solution, and its LS’~~,~ rose to 4.3 (R in figure). The relatively small change in S20,Wwhen 0.2 M KC1 was employed at pH’s 3.5 and 5.4 indicated that even at these pH values the primary charge effect was largely repressed in 0.1 M KCl.
3
4
5
6
7
8
PH
FIG. 1. Effect of pH, urea, and mercaptoethanol on the sedimentation of horse carbon monoxide hemoglobin (0.7%). R indicates value for ,920 obtained at pH 7.5 after reversal of dissociation in 0.1 M KC1 at pH 3.5. Solvents: n 0.1 M KC1 0 0.2 M KC1 X 0.1 M KCI-1.4 M mercaptoethanol 0 0.1 M KCl-4 M urea 0 0.1 M KC1-4 M urea-l.4 M mercaptoethanol
344
GUTTER,
SOBER
AND
PETERSON
Below pH 5.0, in the presence of urea, measurements were not made because of rapid darkening of the solution. In the mixture of urea and mercaptoethanol, at pH 5.2 pink clumps of gelatinous precipitate appeared, and an S20,w of 1.2 was obtained for the clear, straw-colored, supernatant material. When mercaptoethanol alone was used, this bright pink precipitate did not appear until the pH was lowered to 4.2, and ultracentrifugal analysis at this pH revealed two components with sedimentation coefficients of 1.4 and about 25. Most of the color was associated with the heavier component, which appeared as a rather broad and polydisperse peak. This heavy material was recovered as a gel on the floor of the ultracentrifuge cell; the remaining solution contained only the S20,w 1.4 component. After dialysis of the supernatant against several changes of 0.1 M KC1 to remove the mercaptoethanol, the pH was 5.2 and an S20,w of 2.5 was obtained, indicating a partial reversal of the mercaptoethanol effect. No precipitation occurred during the dialysis. From Fig. 1 it can be seen that the reduction of the sedimentation constant of horse hemoglobin at low pH was increased markedly by the presence of either mercaptoethanol or urea, and that a combination of the two was most effective. A suggestion was made in a previous report (1) that a dissociation-association equilibrium exists between subunits of the hemoglobin molecule, which, in the presence of mercaptoethanol or urea, is shifted in the direction of more extensive dissociation. Reichmann and Colvin (7) later discussed a dissociation of horse hemoglobin into four subunits at pH 1.5-2.5 in 0.05 M NaCI, and Shavit and Breuer (8) assumed that separation of electrophoretic components of human hemoglobin at low ionic strengths and high temperatures was due to the TABLE
Concentration Protein
concentration
I
Dependence of Sedimentation
Constant
of
Solvent
PH
Horse COHb S,a.w
x
g./lOO ml.
0.10 0.35 0.70 1.25
7.12 7.12 7.12 7.12
0.10 0.35 0.70 1.25
7.20 7.24 7.23 7.24
0.029
M
K2HPO&).O14
0.2 M KC14
M urea
M
KHzPO(
4.60 4.56 4.40 4.26 3.11 3.21 3.24 3.32
loI*
SEDIMENTATION
OF
CARBON
MONOXIDE
HEMOGLOBIN
345
dissociation of hemoglobin into several units. The latter workers were able to demonstrate that the temperature effects were fully reversible and that equilibrium was established within a few hours. The data in Table I, obtained in studies of the concentration dependence of the s 20,Wof horse hemoglobin in 4 M urea, at neutral pH, support such a concept. In the absence of urea, using pH 7.1,O.l P, potassium phosphate buffer, the SzO,Wdecreased with increasing protein concentration. This agreed with the earlier findings of Kegeles and Gutter (9) concerning the concentration dependence of the Szo,W of human COHb in this solvent. However, in 0.2 M KC14 M urea at nearly the same pH, the Szo,W increased with increasing protein concentration. Only a single boundary was seen in the ultracentrifuge. Since, in a system undergoing reversible dissociation, the law of mass action predicts that an increase in concentration will result in decreased dissociation, one can interpret these observations as evidence for such an equilibrium. The observed increase in &o,~ with increase in protein concentration in the presence of urea is the net result of the Mass Law effect and the opposing effect described above, which probably accounts for its low magnitude. SUMMARY
1. The effect of mercaptoethanol, urea, and their combination upon the sedimentation rate of horse CO-hemoglobin has been determined at pH values of 4.0-7.2. 2. Evidence is presented which supports the theory of a dissociationassociation equilibrium between hemoglobin subunits. REFERENCES 1. GUTTER, F. J., SOBER, H. A., AND PETERSON, E. A., Arch. Biochem. Biophys. 62, 427 (1956). 2. STEINHARDT, J., J. Biol. Chem. 133, 543 (1938). 3. PICKELS, E. G., HARRINGTON, W. F., AND SCHACHMAN, H. II., Proc. N&l. Acad. Sci. U. S. 36, 943 (1952). 4. WAUGH, D. F., AND YPHANTIS, D. A., Rev. Sci. Instr. 23, 609 (1952). 5. BIANCHERIA, A., AND KEGELES, G., J. Am. Chem. Sot. 76, 3737 (1954). 6. FIELD, E. O., AND O’BRIEN, J. R. P., Biochem. J. 60, 656 (1955). 7. REICHMANN, M. E., AND COLVIN, J. R., Can. J. Chem. 34,411 (1956). 8. SHAVIT, N., AND BREUER, M., Biochim. et Biophys. Acta 18, 241 (1955). 9. KEGELES, G., AND GUTTER, F. J., J. Am. Chem. Sot. 73, 3770 (1951).
OF
BIOCHEMIBTRY
AND
BIOPHYSICS
71, 342-345 (1957)
Sedimentation Behavior of Horse Carbon Monoxide Hemoglobin at Low pH in the Presence of Mercaptoethanol and Urea Frederick
J. Gutter,
Herbert
A. Sober and Elbert A. Peterson
From the Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Public Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland Received January
11, 1957
In a previous communication (1) the effect of mercaptoethanol and urea upon the dissociation of hemoglobin between pH 5.8 and 6.6 was reported. The present study compares the effect of pH, at values ranging from 4.0 to 7.2, on the sedimentation of horse hemoglobin in the presence of mercaptoethanol, urea, and their combination, with that observed over a similar pH range in potassium chloride solution alone. METHODS
AND MATERIALS
Horse carbon monoxide hemoglobin (COHb) was prepared by ammonium sulfate fractionation, as in Ref. (l), except for precipitation by 90% saturated rather than 52% saturated ammonium sulfate. The preparation was lyophilized and stored in the cold in an atmosphere of carbon monoxide, and was essentially homogeneous in the analytical ultracentrifuge as well as in moving-boundary electrophoresis at pH 8.6. Baker’s Analyzed Reagent KCl, 2-mercaptoethanol (Eastman Organic Chemicals), and Merckurea (reagent grade, giving a negative Nessler’s test) were employed. Hemoglobin solutions were prepared at a concentration of 0.7% (W/V) in 0.1 M KCI, 0.1 M KC14 M urea, 0.1 M KCl-1.4 M mercaptoethanol, or 0.1 M KC14 1M urea-l.4 M mercaptoethanol. As demonstrated by Steinhardt (a), and corroborated in this study, no further reduction in sedimentation constant was obtained by increasing the urea concentration from 4.0 to 7.5 M at neutral PH. After adjustment with 0.05 N HCl to the appropriate pH, the solutions were dialyzed for 24 hr. at 5°C. on a rocking dialyzer against the same solvent, similarly adjusted. The Spinco synthetic boundary cell (3), and model E analytical ultracentrifuge were used for all measurements. Rotor speed was 59,780 r.p.m. Photographic con342
SEDIMENTATION
OF CARBON MONOXIDE
HEMOGLOBIN
343
ditions, the method of measuring sedimentation constants (SN,~), and the corrections for viscosity, density, temperature, and solution in water have been described (1). The question of the validity of the correction of sedimentation coefficients for viscosity and density in multicomponent systems, according to the Svedberg equation, was considered in the same paper. Correction was also made for the adiabatic cooling of the ultracentrifuge rotor (4, 5). All ultracentrifuge runs were made at room temperature. RESULTS
AND DISCUSSION
The data obtained are shown in Fig. 1. There is good agreement between our results for horse COHb in KC1 alone and those of Field and O’Brien (6) for dissociation of human hemoglobin at low pH. At the lowest pH studied, 3.5, an S20,Wof 2.9 was obtained in this solvent. When the pH was subsequently raised to 7.5, only about 40% of the hemoglobin remained in solution, and its LS’~~,~ rose to 4.3 (R in figure). The relatively small change in S20,Wwhen 0.2 M KC1 was employed at pH’s 3.5 and 5.4 indicated that even at these pH values the primary charge effect was largely repressed in 0.1 M KCl.
3
4
5
6
7
8
PH
FIG. 1. Effect of pH, urea, and mercaptoethanol on the sedimentation of horse carbon monoxide hemoglobin (0.7%). R indicates value for ,920 obtained at pH 7.5 after reversal of dissociation in 0.1 M KC1 at pH 3.5. Solvents: n 0.1 M KC1 0 0.2 M KC1 X 0.1 M KCI-1.4 M mercaptoethanol 0 0.1 M KCl-4 M urea 0 0.1 M KC1-4 M urea-l.4 M mercaptoethanol
344
GUTTER,
SOBER
AND
PETERSON
Below pH 5.0, in the presence of urea, measurements were not made because of rapid darkening of the solution. In the mixture of urea and mercaptoethanol, at pH 5.2 pink clumps of gelatinous precipitate appeared, and an S20,w of 1.2 was obtained for the clear, straw-colored, supernatant material. When mercaptoethanol alone was used, this bright pink precipitate did not appear until the pH was lowered to 4.2, and ultracentrifugal analysis at this pH revealed two components with sedimentation coefficients of 1.4 and about 25. Most of the color was associated with the heavier component, which appeared as a rather broad and polydisperse peak. This heavy material was recovered as a gel on the floor of the ultracentrifuge cell; the remaining solution contained only the S20,w 1.4 component. After dialysis of the supernatant against several changes of 0.1 M KC1 to remove the mercaptoethanol, the pH was 5.2 and an S20,w of 2.5 was obtained, indicating a partial reversal of the mercaptoethanol effect. No precipitation occurred during the dialysis. From Fig. 1 it can be seen that the reduction of the sedimentation constant of horse hemoglobin at low pH was increased markedly by the presence of either mercaptoethanol or urea, and that a combination of the two was most effective. A suggestion was made in a previous report (1) that a dissociation-association equilibrium exists between subunits of the hemoglobin molecule, which, in the presence of mercaptoethanol or urea, is shifted in the direction of more extensive dissociation. Reichmann and Colvin (7) later discussed a dissociation of horse hemoglobin into four subunits at pH 1.5-2.5 in 0.05 M NaCI, and Shavit and Breuer (8) assumed that separation of electrophoretic components of human hemoglobin at low ionic strengths and high temperatures was due to the TABLE
Concentration Protein
concentration
I
Dependence of Sedimentation
Constant
of
Solvent
PH
Horse COHb S,a.w
x
g./lOO ml.
0.10 0.35 0.70 1.25
7.12 7.12 7.12 7.12
0.10 0.35 0.70 1.25
7.20 7.24 7.23 7.24
0.029
M
K2HPO&).O14
0.2 M KC14
M urea
M
KHzPO(
4.60 4.56 4.40 4.26 3.11 3.21 3.24 3.32
loI*
SEDIMENTATION
OF
CARBON
MONOXIDE
HEMOGLOBIN
345
dissociation of hemoglobin into several units. The latter workers were able to demonstrate that the temperature effects were fully reversible and that equilibrium was established within a few hours. The data in Table I, obtained in studies of the concentration dependence of the s 20,Wof horse hemoglobin in 4 M urea, at neutral pH, support such a concept. In the absence of urea, using pH 7.1,O.l P, potassium phosphate buffer, the SzO,Wdecreased with increasing protein concentration. This agreed with the earlier findings of Kegeles and Gutter (9) concerning the concentration dependence of the Szo,W of human COHb in this solvent. However, in 0.2 M KC14 M urea at nearly the same pH, the Szo,W increased with increasing protein concentration. Only a single boundary was seen in the ultracentrifuge. Since, in a system undergoing reversible dissociation, the law of mass action predicts that an increase in concentration will result in decreased dissociation, one can interpret these observations as evidence for such an equilibrium. The observed increase in &o,~ with increase in protein concentration in the presence of urea is the net result of the Mass Law effect and the opposing effect described above, which probably accounts for its low magnitude. SUMMARY
1. The effect of mercaptoethanol, urea, and their combination upon the sedimentation rate of horse CO-hemoglobin has been determined at pH values of 4.0-7.2. 2. Evidence is presented which supports the theory of a dissociationassociation equilibrium between hemoglobin subunits. REFERENCES 1. GUTTER, F. J., SOBER, H. A., AND PETERSON, E. A., Arch. Biochem. Biophys. 62, 427 (1956). 2. STEINHARDT, J., J. Biol. Chem. 133, 543 (1938). 3. PICKELS, E. G., HARRINGTON, W. F., AND SCHACHMAN, H. II., Proc. N&l. Acad. Sci. U. S. 36, 943 (1952). 4. WAUGH, D. F., AND YPHANTIS, D. A., Rev. Sci. Instr. 23, 609 (1952). 5. BIANCHERIA, A., AND KEGELES, G., J. Am. Chem. Sot. 76, 3737 (1954). 6. FIELD, E. O., AND O’BRIEN, J. R. P., Biochem. J. 60, 656 (1955). 7. REICHMANN, M. E., AND COLVIN, J. R., Can. J. Chem. 34,411 (1956). 8. SHAVIT, N., AND BREUER, M., Biochim. et Biophys. Acta 18, 241 (1955). 9. KEGELES, G., AND GUTTER, F. J., J. Am. Chem. Sot. 73, 3770 (1951).