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The carbon monoxide Bohr effect in hemoglobin from Thunnus thynnus
ARCHIVES
OF
BIOCHEMISTRY
AND
The Carbon
BIOPHYSICS
Monoxide
114, 1!%-1% (1966)
Bohr Effect in Hemoglobin
from
Thonnus thynnus MAURIZIO
BRUNORI
Institute of ~~oche~~stry, U~~ver~ty of Rome, Rome, ftdy Received November 5, 1965 The carbon monoxide Bohr effect of Thunnus thynnus hemoglobin was studied by the method of differential titrations. The results can be fitted by the simple model usually applied to mammalian hemoglobins, which aesumes the presence of only two ligated-linked acid groups, one for the acid and one for the alkaline part of the effect. The values of Apg are considerably larger than those usually found in mammalian hemoglobins, being equal to 2.20 for the alkaline group and 1.20 for the acid group. The mean pK values for the two groups are consistent with the view that the two linked acid groups are a carboxyl in the acid range and an imidazole or an a-amino in the alkaline region.
As part of a series of studies on the Bohr effect of different hemoglobins (C-5), undertaken to elucidate the mechanism of heterotropic linkage phenomena in macromolecules, we decided to investigate the behavior of hemoglobin from T~unnu~ ~~~n~~. Besides provid~g the first detailed analysis of the Bohr effect in a salt water fish hemoglobin, this study is of particular interest because the protein involved shows a very peculiar behavior. In fact the shape of the O2 equilibrium curve changes very much with pH, the value of n dropping from about 3 to less than 1 in going from pH 9 to 6 (6). In spite of this, however, the Bohr effect, i.e., the change in the affinity for the ligand with pH, can still be described by the general linkage equations (7) if the median ligand concentration (log p,J is used instead of the ligand concentration corresponding to half saturation (log ~$4). The interrelationship between the change in log p, with. pH and the difference in proton binding capacity accompanying the combination with the ligand is given by the equation
alog% = AH”. ~PH
(1)
Of course, the value of pm can only be established when complete saturation with the ligand can be reached. Since Thunnus thynnus hemoglobin at a pH lower than 6.5 is not saturated with oxygen, even when the partial pressure is 1 atmosphere (6), we have used carbon monoxide as a ligand, and we have investigated the Bohr effect by the differential titration method (8). METHODS The hemoglobin was prepared from the blood of ~~~~~~~ ~~~~~~1 and crystallized from about sulfate according to the method of Rossi-Fanelli and Antonini (9). Because the dissolved crystals contained about 30% of the heme iron in the ferric state, the hemoglobin solution was reduced by an enzymic method (10) and then dialyzed extensively versus double-distilled, boiled water under a stream of argon. After the dialysis, the hemoglobin was deoxygenated in a tonometer and stored as the deoxy derivative. The hemoglobin concentration in heme equivalents was evaluated spectrophotometrically using G,,M = 13.9 at X = $38 rnp for the carbon monoxide derivative (11). The hemoglobin preparation ~88 homogeneous in the ultra~entrifuge, the sedimentation coca1The blood from Thunnus thynnus was kindly supplied by Dr. Mario Molinaro of the Institute of Zoology, University of Palermo. 195
196
BRUNORI
Gent of the carbon monoxide derivative being very similar to that of mammalian hemoglobins (&o,~ = 4.4 at a protein concentration of 6 mg per milliliter). Some 0%equilibrium experiments performed on this material were in excellent agreement with the results of Rossi-Fanelli and Antonini (6). Direct and differential titrations were performed in 0.25 M NaCl at 20” f O.l”C by using the technique previously described (8). The direct hydrogen ion titration of unliganded hemoglobin was nerformed under a stream of purified argon; that of the carbon monoxide derivative was carried out in a closed system under an atmosphere of pure CO. In both cases carefully deoxygenated reagents were used. In each experiment the same solution was titrated forward and backward with 5 X lo-* M NaOH or HCl. RESULTS
The results of the direct hydrogen ion titrations of Hb and HbCO are reported in Fig. 1 and Table I; they are based on the convention that the point of zero net charge for the unliganded derivative is equal to pH 7.0 at 2O’C. As a matter of
fact, although no accurate measurements have been made to establish the isoionic point of Hb, by mixing unliganded hemoglobin with NaCl the re~lting pH was found to be very near to 7 in three separate experiments. The relative position of the direct titration curve for HbCO in respect to that for Hb was establ~hed by a series of differential measurements, in which both the ApH and AH+ values accompanying the binding of the ligand were determined. Such results are reported in Fig. 2, in which are reproduced as smooth curves the direct hydrogen ion titrations for the two derivatives reported in Fig. 1. The values of AH+, the difference between proton bound by Hb and HbCO at different pH values, can be obtained directly from Fig. 2, and are reported in Table I. DISCUSSION
According to Eq. (l), the integral fAH+dpH should give the change in the log
PH 5
6
7
8
9
10
4
5
6
7
8
9
-5 -4 -3 g-2 z-1 2 5
0
0
g1 2 3 4
10
Fro. 1. Direct hydroge inon titration curves for Hb and HbCO from T~~~~~s ~~~~us in 0.25 M NaCl and 26°C. The abscissa at the top applies to HbCO and that at the bottom to Hb. The closed and open circles indicate the forward and backward titrations, respectively.
BOHR EFFECT
5
IN TUNA
6
7
197
FISH Hb
8
9
10
pw FIG. 2. The direct hydrogen ion titration curves for Hb and HbCO are reported as smooth lines (solid and dashed) reproduced from Fig. 1. The points indicate the results of the differential titration measurements, with the following notations: l + 0, change in pH ~compan~ng the binding of CO by Hb; (>, corresponding values of AHi.
of the median ligand concentration with pH. As already stated, the value of p,,, is only accessible to direct experimental determination if saturation is reached at some attainable value of the ligand concentration. It was for dhis reason, in view of the results obtained bv Rossi-Fanelli and Antonini on the O2 equilibrium of the hemoglobin (6), that we used CO as a ligand. We have shown that at 1 atmosphere Thunnus thynnus hemoglob~ is fully saturated with this ligand even at pH 5.1. Figure 3 shows the Bohr effect in terms of log p, for Thunnus thynnus hemoglobin as calculated from differential measurements. It will be seen that the general shape of the curve is very similar to that reported for other hemog~ob~s, showing a region of minimum ligand aihnity at pH ~6 and an increase on both the acid and the alkaline sides. Moreover, the Bohr effect curve for Tuna hemoglobin can be described quantit,atively by the simple model applied to mammali~ hemoglob~s (7), which assumes
the existence of only two ligand linked acid groups per heme, one responsible for the normal (or alkaline), and the other for the reverse (or acid) Bohr effects. A satisfactory fit of the data is obtained with the values of the constants reported in Table II. These pK values for the two ionizable groups are consistent with the view, recently suggested for the case of human and horse hemoglobins (l), that the groups responsible for the reverse Bohr effect are earboxyl groups, and those responsible for the normal effect are imidazole or a-amino groups. On the other hand, the pK changes for both groups produced by the binding of carbon monoxide are by far the largest every reported in the literature for any hemoglobin. Thus the maximum value of AH+ (at pH -7.5) is equal to 0.86 H+ per heme as compared to the value of 0.56 found for human hemoglobin (1). Although no complete gasometric measurements are available with which to compare these results, nevertheless over the range in which they are
198
BRUNORI
0.5
4
5
6
8
7
10
9
FIG. 3. Change in median ligand concentration (log p,) with pH (Bohr effect) in the binding of carbon monoxide by hemoglobin from Thunnus thynnus, in 0.25 M NaCl and 20°C. 0, values calculated from the change in AH+ with pH according to equation (1); solid line is calculated according to the constants given in Table II. The values reported on the ordinates are wholly arbitrary.
TABLE
TABLE
I
HYDROGEN ION BWJSD PER HEME BY THUNNUS TEYNNUS CARBON MONOXIDE AND DEOXYHEMOGLOBINS~ AH+@+w -%@J
PH
%bCO
5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4
+4.94
-1.84 -2.12 -2.34 -2.58 -2.80
+0.75 +0.38 0 -0.37 -0.68 -1.02 -1.36 -1.70 -2.05 -2.40
8.6
-3.04
-2.74
f0.86 +o.s2 +0.76 +0.64 +0.53 to.40 +0.30
8.8 9.0 9.2
-3.30 -3.64 -4.00
-3.08 -3.50 -
+0.22 -j-o.14 -
+4.10 +3.37 +2.75 $2.18 +1.62 +1.09 +0.59
+0.10 -0.39 -0.80 -1.18 -1.54
+4.32 +3.56 +2.9s +2.52 +2.11
f1.78 +1.43 +1.08
-0.62 -0.54 -0.39 -0.23 -0.07 +0.16 f0.34 +0.49 +0.65 -to.77 +o.so
+o.l31
a Me~urements in 0.25 M NaCl, at 20” C. Protein concentration, 0.5%.
II
CONSTANTS FOR THE CARBON MONOXIDELINKED ACID GROUPS IN HEMOGLOBIN FROM Tmmaw TEYNNUS, IN 0.25 M NaCl AND 20°C Constant
Hb
HbCO
APK
PK~ PK~
4.60 8.28
5.80 6.08
2.20
1.20
complete the oxygen results are in agreement with the differential titrations. In conclusion these results bring out once again that the Bohr effect is a general phenomenon which exhibits a pattern colon in all hemoglobins so far studied, in spite of significant quantzitative variations which undoubtedly reflect specific structural differences. ACKNOWLEDGMENT It is a pleasure to express sincere thanks to Professors Eraldo Antonini and Jeffries Wyman for their helpful suggestions and encouragement. REFERENCES 1. ANTONINI, E., WYMAN, J., BRUNORI, M., FRONTICELLX, C., BUCCI, E., AND ROSSIFANELLI, A., J. Bid. Chem. 940, 1096
(1965).
BOHR
EFFECT
2. ANTONINI, E., WYMAN, J., BELLELLI, L., RUMEN, N., AND SINISCAIEO, M., Arch. Biochem. Biophys. 106, 404 (1964). 3. ANTONINI,, E., WYMAN, J., BRUNORI, M., FRONTI~ELLI, C., Bucc~, E., REICHLIN, M., AND ROSSI-FANELLI, A., Arch. Biochem. Biophys. 108, 569 (1964). 4. ANTONINI. E., WYMAN, J., BUCCI, E., FRONTICELLI, C., BRKJNORI, M., REICHLIN, M., AND ROSSI-FANELLI, A., Biochim. Biophys. Acta 104, 160 (1965). 5. SMITH, D. B., BRUNORI, M., ANTONINI, E., AND WYMAN, J., Arch. Biochem. Biophys. 113, 725 (1966).
IN
TUNA
FISH
Hb
199
6. ROSH-FANELLI, A., AND ANTONINI, E., Nature 186, 895 (1960). 7. WYMAN, J., Advan. Protein Chem. 19, 223 (1964). 8. ANTONINI, E., WYMAN, J., BRUNORI, M., BUCCI, E., FRONTICELLI, C., AND ROSSIFANELLI, A., J. Biol. Chem. 238, 2950 (1963). 9. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 68, 498 (1955). 10. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 77, 478 (1958). 11. BUCCI, E., AND FRONTICELLI, C., Boll. Sot. It. Biol. Sperim. 37, 1765 (1961).
OF
BIOCHEMISTRY
AND
The Carbon
BIOPHYSICS
Monoxide
114, 1!%-1% (1966)
Bohr Effect in Hemoglobin
from
Thonnus thynnus MAURIZIO
BRUNORI
Institute of ~~oche~~stry, U~~ver~ty of Rome, Rome, ftdy Received November 5, 1965 The carbon monoxide Bohr effect of Thunnus thynnus hemoglobin was studied by the method of differential titrations. The results can be fitted by the simple model usually applied to mammalian hemoglobins, which aesumes the presence of only two ligated-linked acid groups, one for the acid and one for the alkaline part of the effect. The values of Apg are considerably larger than those usually found in mammalian hemoglobins, being equal to 2.20 for the alkaline group and 1.20 for the acid group. The mean pK values for the two groups are consistent with the view that the two linked acid groups are a carboxyl in the acid range and an imidazole or an a-amino in the alkaline region.
As part of a series of studies on the Bohr effect of different hemoglobins (C-5), undertaken to elucidate the mechanism of heterotropic linkage phenomena in macromolecules, we decided to investigate the behavior of hemoglobin from T~unnu~ ~~~n~~. Besides provid~g the first detailed analysis of the Bohr effect in a salt water fish hemoglobin, this study is of particular interest because the protein involved shows a very peculiar behavior. In fact the shape of the O2 equilibrium curve changes very much with pH, the value of n dropping from about 3 to less than 1 in going from pH 9 to 6 (6). In spite of this, however, the Bohr effect, i.e., the change in the affinity for the ligand with pH, can still be described by the general linkage equations (7) if the median ligand concentration (log p,J is used instead of the ligand concentration corresponding to half saturation (log ~$4). The interrelationship between the change in log p, with. pH and the difference in proton binding capacity accompanying the combination with the ligand is given by the equation
alog% = AH”. ~PH
(1)
Of course, the value of pm can only be established when complete saturation with the ligand can be reached. Since Thunnus thynnus hemoglobin at a pH lower than 6.5 is not saturated with oxygen, even when the partial pressure is 1 atmosphere (6), we have used carbon monoxide as a ligand, and we have investigated the Bohr effect by the differential titration method (8). METHODS The hemoglobin was prepared from the blood of ~~~~~~~ ~~~~~~1 and crystallized from about sulfate according to the method of Rossi-Fanelli and Antonini (9). Because the dissolved crystals contained about 30% of the heme iron in the ferric state, the hemoglobin solution was reduced by an enzymic method (10) and then dialyzed extensively versus double-distilled, boiled water under a stream of argon. After the dialysis, the hemoglobin was deoxygenated in a tonometer and stored as the deoxy derivative. The hemoglobin concentration in heme equivalents was evaluated spectrophotometrically using G,,M = 13.9 at X = $38 rnp for the carbon monoxide derivative (11). The hemoglobin preparation ~88 homogeneous in the ultra~entrifuge, the sedimentation coca1The blood from Thunnus thynnus was kindly supplied by Dr. Mario Molinaro of the Institute of Zoology, University of Palermo. 195
196
BRUNORI
Gent of the carbon monoxide derivative being very similar to that of mammalian hemoglobins (&o,~ = 4.4 at a protein concentration of 6 mg per milliliter). Some 0%equilibrium experiments performed on this material were in excellent agreement with the results of Rossi-Fanelli and Antonini (6). Direct and differential titrations were performed in 0.25 M NaCl at 20” f O.l”C by using the technique previously described (8). The direct hydrogen ion titration of unliganded hemoglobin was nerformed under a stream of purified argon; that of the carbon monoxide derivative was carried out in a closed system under an atmosphere of pure CO. In both cases carefully deoxygenated reagents were used. In each experiment the same solution was titrated forward and backward with 5 X lo-* M NaOH or HCl. RESULTS
The results of the direct hydrogen ion titrations of Hb and HbCO are reported in Fig. 1 and Table I; they are based on the convention that the point of zero net charge for the unliganded derivative is equal to pH 7.0 at 2O’C. As a matter of
fact, although no accurate measurements have been made to establish the isoionic point of Hb, by mixing unliganded hemoglobin with NaCl the re~lting pH was found to be very near to 7 in three separate experiments. The relative position of the direct titration curve for HbCO in respect to that for Hb was establ~hed by a series of differential measurements, in which both the ApH and AH+ values accompanying the binding of the ligand were determined. Such results are reported in Fig. 2, in which are reproduced as smooth curves the direct hydrogen ion titrations for the two derivatives reported in Fig. 1. The values of AH+, the difference between proton bound by Hb and HbCO at different pH values, can be obtained directly from Fig. 2, and are reported in Table I. DISCUSSION
According to Eq. (l), the integral fAH+dpH should give the change in the log
PH 5
6
7
8
9
10
4
5
6
7
8
9
-5 -4 -3 g-2 z-1 2 5
0
0
g1 2 3 4
10
Fro. 1. Direct hydroge inon titration curves for Hb and HbCO from T~~~~~s ~~~~us in 0.25 M NaCl and 26°C. The abscissa at the top applies to HbCO and that at the bottom to Hb. The closed and open circles indicate the forward and backward titrations, respectively.
BOHR EFFECT
5
IN TUNA
6
7
197
FISH Hb
8
9
10
pw FIG. 2. The direct hydrogen ion titration curves for Hb and HbCO are reported as smooth lines (solid and dashed) reproduced from Fig. 1. The points indicate the results of the differential titration measurements, with the following notations: l + 0, change in pH ~compan~ng the binding of CO by Hb; (>, corresponding values of AHi.
of the median ligand concentration with pH. As already stated, the value of p,,, is only accessible to direct experimental determination if saturation is reached at some attainable value of the ligand concentration. It was for dhis reason, in view of the results obtained bv Rossi-Fanelli and Antonini on the O2 equilibrium of the hemoglobin (6), that we used CO as a ligand. We have shown that at 1 atmosphere Thunnus thynnus hemoglob~ is fully saturated with this ligand even at pH 5.1. Figure 3 shows the Bohr effect in terms of log p, for Thunnus thynnus hemoglobin as calculated from differential measurements. It will be seen that the general shape of the curve is very similar to that reported for other hemog~ob~s, showing a region of minimum ligand aihnity at pH ~6 and an increase on both the acid and the alkaline sides. Moreover, the Bohr effect curve for Tuna hemoglobin can be described quantit,atively by the simple model applied to mammali~ hemoglob~s (7), which assumes
the existence of only two ligand linked acid groups per heme, one responsible for the normal (or alkaline), and the other for the reverse (or acid) Bohr effects. A satisfactory fit of the data is obtained with the values of the constants reported in Table II. These pK values for the two ionizable groups are consistent with the view, recently suggested for the case of human and horse hemoglobins (l), that the groups responsible for the reverse Bohr effect are earboxyl groups, and those responsible for the normal effect are imidazole or a-amino groups. On the other hand, the pK changes for both groups produced by the binding of carbon monoxide are by far the largest every reported in the literature for any hemoglobin. Thus the maximum value of AH+ (at pH -7.5) is equal to 0.86 H+ per heme as compared to the value of 0.56 found for human hemoglobin (1). Although no complete gasometric measurements are available with which to compare these results, nevertheless over the range in which they are
198
BRUNORI
0.5
4
5
6
8
7
10
9
FIG. 3. Change in median ligand concentration (log p,) with pH (Bohr effect) in the binding of carbon monoxide by hemoglobin from Thunnus thynnus, in 0.25 M NaCl and 20°C. 0, values calculated from the change in AH+ with pH according to equation (1); solid line is calculated according to the constants given in Table II. The values reported on the ordinates are wholly arbitrary.
TABLE
TABLE
I
HYDROGEN ION BWJSD PER HEME BY THUNNUS TEYNNUS CARBON MONOXIDE AND DEOXYHEMOGLOBINS~ AH+@+w -%@J
PH
%bCO
5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4
+4.94
-1.84 -2.12 -2.34 -2.58 -2.80
+0.75 +0.38 0 -0.37 -0.68 -1.02 -1.36 -1.70 -2.05 -2.40
8.6
-3.04
-2.74
f0.86 +o.s2 +0.76 +0.64 +0.53 to.40 +0.30
8.8 9.0 9.2
-3.30 -3.64 -4.00
-3.08 -3.50 -
+0.22 -j-o.14 -
+4.10 +3.37 +2.75 $2.18 +1.62 +1.09 +0.59
+0.10 -0.39 -0.80 -1.18 -1.54
+4.32 +3.56 +2.9s +2.52 +2.11
f1.78 +1.43 +1.08
-0.62 -0.54 -0.39 -0.23 -0.07 +0.16 f0.34 +0.49 +0.65 -to.77 +o.so
+o.l31
a Me~urements in 0.25 M NaCl, at 20” C. Protein concentration, 0.5%.
II
CONSTANTS FOR THE CARBON MONOXIDELINKED ACID GROUPS IN HEMOGLOBIN FROM Tmmaw TEYNNUS, IN 0.25 M NaCl AND 20°C Constant
Hb
HbCO
APK
PK~ PK~
4.60 8.28
5.80 6.08
2.20
1.20
complete the oxygen results are in agreement with the differential titrations. In conclusion these results bring out once again that the Bohr effect is a general phenomenon which exhibits a pattern colon in all hemoglobins so far studied, in spite of significant quantzitative variations which undoubtedly reflect specific structural differences. ACKNOWLEDGMENT It is a pleasure to express sincere thanks to Professors Eraldo Antonini and Jeffries Wyman for their helpful suggestions and encouragement. REFERENCES 1. ANTONINI, E., WYMAN, J., BRUNORI, M., FRONTICELLX, C., BUCCI, E., AND ROSSIFANELLI, A., J. Bid. Chem. 940, 1096
(1965).
BOHR
EFFECT
2. ANTONINI, E., WYMAN, J., BELLELLI, L., RUMEN, N., AND SINISCAIEO, M., Arch. Biochem. Biophys. 106, 404 (1964). 3. ANTONINI,, E., WYMAN, J., BRUNORI, M., FRONTI~ELLI, C., Bucc~, E., REICHLIN, M., AND ROSSI-FANELLI, A., Arch. Biochem. Biophys. 108, 569 (1964). 4. ANTONINI. E., WYMAN, J., BUCCI, E., FRONTICELLI, C., BRKJNORI, M., REICHLIN, M., AND ROSSI-FANELLI, A., Biochim. Biophys. Acta 104, 160 (1965). 5. SMITH, D. B., BRUNORI, M., ANTONINI, E., AND WYMAN, J., Arch. Biochem. Biophys. 113, 725 (1966).
IN
TUNA
FISH
Hb
199
6. ROSH-FANELLI, A., AND ANTONINI, E., Nature 186, 895 (1960). 7. WYMAN, J., Advan. Protein Chem. 19, 223 (1964). 8. ANTONINI, E., WYMAN, J., BRUNORI, M., BUCCI, E., FRONTICELLI, C., AND ROSSIFANELLI, A., J. Biol. Chem. 238, 2950 (1963). 9. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 68, 498 (1955). 10. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 77, 478 (1958). 11. BUCCI, E., AND FRONTICELLI, C., Boll. Sot. It. Biol. Sperim. 37, 1765 (1961).