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The effect of oxygen, carbon dioxide, pH and cyanate on the binding of 2,3-diphosphoglycerate to human hemoglobin
Vol. 44, No. 6,1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
THE EFFECT OF OXYGEN~ CARBON DIOXIDE~ pH AND CYANATE ON THE BINDING OF 2~3-DIPHOSPHOGLYCERATE TO HUMAN HEMOGLOBIN
P.R.B. Caldwell~ R.L. Nagel and E.R. Jaff~ Departments of Medicine College of Physicians and Surgeons and Albert Einstein College of Medicine New Yorkj N.Y.
Received July19, 1971 SUMMARY - The binding of 2~3-diphosphoglycerate (2~3-DPG) to human hemoglobin was studied by equilibrium dialysis in a tonometer system. The binding of 2~3-DPG was inversely related to both oxygen saturation and pH within the physiologic ranges. Carbon dioxide and cyanate inhibited binding~ suggesting that the ~ amino groups of one or more of the N-terminal amino acids of hemoglobin are involved in the binding of 2~3-DPG.
Benesch~ Benesch and Yu (i) demonstrated that 233-DPG binds to completely deoxygenated~ but not to fully oxygenated~ hemoglobin in the presence of 0.i M NaCI.
Since hemoglobin in vivo exists at levels of oxygen
saturation which are intermediate to those studied~ we measured the binding of 233-DPG to hemoglobin over the full range of saturation by equilibrium dialysis in a tonometer system where gas tensions could be controlled precisely. Bunn and Briehl (2) provided data suggesting that the N-terminal amino groups of the beta chains are involved in the binding of 2~3-DPG to hemoglobin.
Since carbon dioxide and cyanate both react specifically with
free ~ amino groups of hemoglobin to form carbamino and carbamylated derivatives 3 respectively (3~4~5)~ we tested the effect of these compounds on the binding of 2~3-DPG to hemoglobin. METHODS Hemolysates~ prepared by a modification of the method of Drabkin (i~6)
1504
Vol. 44, No. 6,1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
from fresh human blood collected in eitrat% were dialyzed against 0.i NaCI for 20 hours using #20 cellulose casing (Union Carbide) which had been previously stretched (7). The dialyzed hemolysate was diluted with 0.05 bis Tris adjusted to the desired pH and salt concentration. dialysis s a %
A stretched
containing 5 ml of the hemoglobin solution~ was placed in a
swirl tonometer (Instrumentation Laboratories~ Inc.) with 25 ml of the same buffer outside the casing.
The tonometer was pre-equilibrated with the
desired oxygen and carbon dioxide mixtures.
2~3-DPG-U-14C (0.3 ~Ci/BMole)
was added to the buffer outside the dialysis sac to give the indicated final concentrations.
Equilibrium as measured by the distribution of radio-
activitywas complete after 5 hours of tonometry. tonometer was maintained at 20°C.
The temperature of the
Analysis of the labelled 2,3-DPG by
paper chromatography showed that the distribution of radioactivity was not altered during tonometry.
Spectrophotometric analyses of the hemoglobin
after tonometry revealed less than 3% methemoglobin.
In parallel studies~
the oxygen saturation obtained under the conditions used for each gas mixture was determined spectrophotometrically.
Binding was calculated from the
measurement of radioactivity remaining in the buffer solution outside the sac at equilibrium.
This concentration represented the free 2,3-DPG.
The
difference between the total cpm added at the beginning and the total epm outside the sac at equilibrium was the sum of the free plus bound 2~3-DPG inside the sac.
In separate studies no binding by the cellulose membran%
itself~ could be detected. during tonometry.
There was no volume change in the sac contents
Carbamylated hemoglobin was prepared in a reaction mixture
of 6 x 10 -4 ~noles of dialyzed hemoglobin and 3 x 10 -2 mmoles of KCNO in 0.05 M bis Tris~ 0.i M NaCI at pH 6.9~ incubated for one hour at 20°C (8). All hemoglobin concentrations are expressed in molarities based on the tetrameric molecular weight of 64j500 g.
RESULTS A preliminary study confirmed the observations of Beneseh~ Benesch and
1505
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Vol. 44, No. 6, 1971
Yu (I) regarding the effect of salt concentration on 2~3-DPG binding to hemoglobin.
A Scatchard plot (9) of these data 3 shown in Figure 13 indicates
that binding approaches a i:I molar stoichiometry in 0.i M NaCI.
At a lower
salt concentration the reaction is complex and probably involves multiple binding sites.
4
Hb IxlO'4 M 0,05 M BIS TRIS 20 °C pH 6,9
'\\ \
BOUND 2~3-DPG 3 FREE 2,3- DPG 2
0
I
" " " o ~ .., °
0
I I 5 IO 15 BOUND 2,3-DPG =10 -4 mmole
Figure io A Scatchard plot (9) of 233-DPG binding to deoxyhemoglobin (6 x 10 -4 rmuoles) at 0.i (0) and 0.01 (0) M NaCI.
,25 ,20 ,15 MOLES 2,5-DPG BOUND MOLES HEMOGLOBIN ,10 ,05
1,2 x 10-4M 0.1 M NaCI 0.05 M BIS TRIS °C
Hb el
70
°
I
7,4
.I
o
7,8
I 8.2
pH.
Figure 2. The effect of pH on 2j3-DPG binding to deoxy-(0) and oxy-(0) hemoglobin.
The effect of pH on the binding of 2j3-DPG to deoxy- and oxyhemoglobin is shown in Figure 2.
These results are consistent with earlier observa1506
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tions (i0) and demonstrate that binding is inversely related to pH at the physiologic level (pH 7.2).
The relation between 2~3-DPG binding and oxygen
saturation at pH 7.2 is depicted in Figure 3. average of two or more determinations. of regression is 0.995.
Each point represents an
The correlation for a linear line
Under the conditions studied there was minimalj
though measurabl% binding of 2~3-DPG to oxyhemoglobin.
.2C Hb 1.2 x IO'*M 0.1 M NaCI 0,05 M DIS TRIS 20 °C
.15
MOLES 2,3-DPG BOUND,to MOLES HEMOGLOBIN .0!
I
I
I
f
25
50
75
I00
OXYGEN SATURATION (%)
Figure 3.
The binding of 2~3-DPG to hemoglobin versus oxygen saturation.
25 10"4M tCI ,2C
BIS TRIS
,15
MOLES 2,5-DPG BOUND MOLES HEMOGLOBIN ,IC
0f
Figure 4. The effect of carbon dioxide (60 ~n Hg) and cyanate in molar excess (50:1) on 2~3-DPG binding to deoxyhemoglobin.
The influence of carbon dioxide and cyanate and their combined effect are illustrated in Figure 4.
There is a 30 per cent reduction in binding
of carbamylated hemoglobin and a 55 per cent reduction in the presence of 60 mm Hg carbon dioxide.
There is no additive effect for the combination
of cyanate and carbon dioxide under these conditions.
1507
At a more physiologic
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
level of carbon dioxide (40 nTm Hg) there is a 25 per cent reduction in 233-DPG binding to hemoglobin. DISCUSSION These results demonstrate the usefulness of the tonometer system for studies of equilibrium dialysis where small differences in gas tension might be expected to have a marked effect on binding. The differential binding of 2j3-DPG to hemoglobin over the physiologic range of oxygen saturation (75-95%) may account 3 in part 3 for the difference in oxygen affinity between arterial and venous blood.
In addition 3 these
observations support the suggestion that oxygen saturation may play a role in the control of erythrocyte content of 233-DPG ~ since binding of 233-DPG might prevent the demonstrated inhibition of diphosphoglycerate mutase by free 233-DPG (ii).
In this regard it should be noted that H+ has the
potential for opposing effects on the erythrocyte content of 2~3-DPG.
An
increase in H+ reduces the synthesis of 2~3-DPG by slowing glycolysis (12) and at the same time 3 by favoring 2~3-DPG binding~ removes its end product inhibition of diphosphoglycerate mutase. The demonstration of specific binding of 233-DPG to deoxyhemoglobin at physiologic salt concentrations (0.i M_)3 confirmed here 3 and its partial inhibition by cyanate and carbon dioxide are consistent with a I:i molar stoichiometry of this binding.
Benesch 3 Benesch and Enoki (13) suggested
that the site of binding might be on the diad axis of syr~netry between the beta chains.
Perutz (14) proposed 3 on the basis of model fitting and the
data of Bunn and Briehl (2) 3 that two of the paired charged groups involved were the ~ amino groups of the N-terminal valine residues in the beta chains. Kilmartin and Rossi-Bernardi (8) have shown that carbon dioxide binds these terminal amino groups.
Moreover 3 the competition of carbon dioxide and
2~3-DPG for these sites has been inferred from the interference of carbon dioxide with the 2~3-DPG effect on the oxygen equilibrium of hemoglobi% as shown recently by Tomita and Riggs (15). The inhibition of 2~3-DPG
1508
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
binding to hemoglobin, per se, by both carbon dioxide and cyanate provides strong evidence that the ~ amino groups participate in the binding site of hemoglobin for 2,3-DPG. ACKNOWLEDGEMENT This work was supported by USPHS Grants # AM-13430, AM-13698, AM-54353 AM-15053, HE-02001, HE-05741, HE-05443 and a grant-in aid from the New York Heart Association.
The authors are Career Scientists of the Health Research
Council of the City of New York.
REFERENCES I. 2. 3. 4. 5. 6o 7. 8. 9. i0. ii. 12. 13.
14. 15.
Benesch, R., R.E. Benesch and C.I. Yu, Proc. Nat. Acad. Sci. 59, 526 (1968). Bunn, H.F. and R.W. Briehl, J. Clin. Invest. 49, 1088 (1970). Stark, G.R. and D.G. Smyth, J. Biol. Chem. 238, 214 (1963). Kilmartin, JoV. and L. Rossi-Bernardi~ Nature. 222, 1243 (1969). Cerami, A. and J.M. Manning, Proe. Nat. Acad. Sci. 683 1180 (1971). Drabkin, D.L., J. Biol. Chem. 164. 703 (1946). Craig~ L.C. Advan. Anal. Chem. Instr. 41, 35 (1965). Kilmartin, J.V. and L. Rossi-Bernardi, NASA SP-188, 73 (1969). Scatchard, G., Ann. N.Y. Acad. Sci. 51, 660 (1949). Beneschj R.E., R. Benesch and C.I. Yu~ Biochem. 8, 2567 (1969). Rose, Z.B., Fed. Proceed. 29, 1105 (1970). Minakami, S. and H. Yoshikawa, J. Biochem. 59, 145 (1966). Benesch, R., R.E. Benesch and Y. Enoki, Proc. Nat. Aead. Sci. 61, 1102 (1968) o Perutz, M.F., Nature 228, 734 (1970). Tomita, S. and A. Rigg% J. Biol. Chem. 246, 547 (1971)o
1509
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
THE EFFECT OF OXYGEN~ CARBON DIOXIDE~ pH AND CYANATE ON THE BINDING OF 2~3-DIPHOSPHOGLYCERATE TO HUMAN HEMOGLOBIN
P.R.B. Caldwell~ R.L. Nagel and E.R. Jaff~ Departments of Medicine College of Physicians and Surgeons and Albert Einstein College of Medicine New Yorkj N.Y.
Received July19, 1971 SUMMARY - The binding of 2~3-diphosphoglycerate (2~3-DPG) to human hemoglobin was studied by equilibrium dialysis in a tonometer system. The binding of 2~3-DPG was inversely related to both oxygen saturation and pH within the physiologic ranges. Carbon dioxide and cyanate inhibited binding~ suggesting that the ~ amino groups of one or more of the N-terminal amino acids of hemoglobin are involved in the binding of 2~3-DPG.
Benesch~ Benesch and Yu (i) demonstrated that 233-DPG binds to completely deoxygenated~ but not to fully oxygenated~ hemoglobin in the presence of 0.i M NaCI.
Since hemoglobin in vivo exists at levels of oxygen
saturation which are intermediate to those studied~ we measured the binding of 233-DPG to hemoglobin over the full range of saturation by equilibrium dialysis in a tonometer system where gas tensions could be controlled precisely. Bunn and Briehl (2) provided data suggesting that the N-terminal amino groups of the beta chains are involved in the binding of 2~3-DPG to hemoglobin.
Since carbon dioxide and cyanate both react specifically with
free ~ amino groups of hemoglobin to form carbamino and carbamylated derivatives 3 respectively (3~4~5)~ we tested the effect of these compounds on the binding of 2~3-DPG to hemoglobin. METHODS Hemolysates~ prepared by a modification of the method of Drabkin (i~6)
1504
Vol. 44, No. 6,1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
from fresh human blood collected in eitrat% were dialyzed against 0.i NaCI for 20 hours using #20 cellulose casing (Union Carbide) which had been previously stretched (7). The dialyzed hemolysate was diluted with 0.05 bis Tris adjusted to the desired pH and salt concentration. dialysis s a %
A stretched
containing 5 ml of the hemoglobin solution~ was placed in a
swirl tonometer (Instrumentation Laboratories~ Inc.) with 25 ml of the same buffer outside the casing.
The tonometer was pre-equilibrated with the
desired oxygen and carbon dioxide mixtures.
2~3-DPG-U-14C (0.3 ~Ci/BMole)
was added to the buffer outside the dialysis sac to give the indicated final concentrations.
Equilibrium as measured by the distribution of radio-
activitywas complete after 5 hours of tonometry. tonometer was maintained at 20°C.
The temperature of the
Analysis of the labelled 2,3-DPG by
paper chromatography showed that the distribution of radioactivity was not altered during tonometry.
Spectrophotometric analyses of the hemoglobin
after tonometry revealed less than 3% methemoglobin.
In parallel studies~
the oxygen saturation obtained under the conditions used for each gas mixture was determined spectrophotometrically.
Binding was calculated from the
measurement of radioactivity remaining in the buffer solution outside the sac at equilibrium.
This concentration represented the free 2,3-DPG.
The
difference between the total cpm added at the beginning and the total epm outside the sac at equilibrium was the sum of the free plus bound 2~3-DPG inside the sac.
In separate studies no binding by the cellulose membran%
itself~ could be detected. during tonometry.
There was no volume change in the sac contents
Carbamylated hemoglobin was prepared in a reaction mixture
of 6 x 10 -4 ~noles of dialyzed hemoglobin and 3 x 10 -2 mmoles of KCNO in 0.05 M bis Tris~ 0.i M NaCI at pH 6.9~ incubated for one hour at 20°C (8). All hemoglobin concentrations are expressed in molarities based on the tetrameric molecular weight of 64j500 g.
RESULTS A preliminary study confirmed the observations of Beneseh~ Benesch and
1505
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Vol. 44, No. 6, 1971
Yu (I) regarding the effect of salt concentration on 2~3-DPG binding to hemoglobin.
A Scatchard plot (9) of these data 3 shown in Figure 13 indicates
that binding approaches a i:I molar stoichiometry in 0.i M NaCI.
At a lower
salt concentration the reaction is complex and probably involves multiple binding sites.
4
Hb IxlO'4 M 0,05 M BIS TRIS 20 °C pH 6,9
'\\ \
BOUND 2~3-DPG 3 FREE 2,3- DPG 2
0
I
" " " o ~ .., °
0
I I 5 IO 15 BOUND 2,3-DPG =10 -4 mmole
Figure io A Scatchard plot (9) of 233-DPG binding to deoxyhemoglobin (6 x 10 -4 rmuoles) at 0.i (0) and 0.01 (0) M NaCI.
,25 ,20 ,15 MOLES 2,5-DPG BOUND MOLES HEMOGLOBIN ,10 ,05
1,2 x 10-4M 0.1 M NaCI 0.05 M BIS TRIS °C
Hb el
70
°
I
7,4
.I
o
7,8
I 8.2
pH.
Figure 2. The effect of pH on 2j3-DPG binding to deoxy-(0) and oxy-(0) hemoglobin.
The effect of pH on the binding of 2j3-DPG to deoxy- and oxyhemoglobin is shown in Figure 2.
These results are consistent with earlier observa1506
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tions (i0) and demonstrate that binding is inversely related to pH at the physiologic level (pH 7.2).
The relation between 2~3-DPG binding and oxygen
saturation at pH 7.2 is depicted in Figure 3. average of two or more determinations. of regression is 0.995.
Each point represents an
The correlation for a linear line
Under the conditions studied there was minimalj
though measurabl% binding of 2~3-DPG to oxyhemoglobin.
.2C Hb 1.2 x IO'*M 0.1 M NaCI 0,05 M DIS TRIS 20 °C
.15
MOLES 2,3-DPG BOUND,to MOLES HEMOGLOBIN .0!
I
I
I
f
25
50
75
I00
OXYGEN SATURATION (%)
Figure 3.
The binding of 2~3-DPG to hemoglobin versus oxygen saturation.
25 10"4M tCI ,2C
BIS TRIS
,15
MOLES 2,5-DPG BOUND MOLES HEMOGLOBIN ,IC
0f
Figure 4. The effect of carbon dioxide (60 ~n Hg) and cyanate in molar excess (50:1) on 2~3-DPG binding to deoxyhemoglobin.
The influence of carbon dioxide and cyanate and their combined effect are illustrated in Figure 4.
There is a 30 per cent reduction in binding
of carbamylated hemoglobin and a 55 per cent reduction in the presence of 60 mm Hg carbon dioxide.
There is no additive effect for the combination
of cyanate and carbon dioxide under these conditions.
1507
At a more physiologic
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
level of carbon dioxide (40 nTm Hg) there is a 25 per cent reduction in 233-DPG binding to hemoglobin. DISCUSSION These results demonstrate the usefulness of the tonometer system for studies of equilibrium dialysis where small differences in gas tension might be expected to have a marked effect on binding. The differential binding of 2j3-DPG to hemoglobin over the physiologic range of oxygen saturation (75-95%) may account 3 in part 3 for the difference in oxygen affinity between arterial and venous blood.
In addition 3 these
observations support the suggestion that oxygen saturation may play a role in the control of erythrocyte content of 233-DPG ~ since binding of 233-DPG might prevent the demonstrated inhibition of diphosphoglycerate mutase by free 233-DPG (ii).
In this regard it should be noted that H+ has the
potential for opposing effects on the erythrocyte content of 2~3-DPG.
An
increase in H+ reduces the synthesis of 2~3-DPG by slowing glycolysis (12) and at the same time 3 by favoring 2~3-DPG binding~ removes its end product inhibition of diphosphoglycerate mutase. The demonstration of specific binding of 233-DPG to deoxyhemoglobin at physiologic salt concentrations (0.i M_)3 confirmed here 3 and its partial inhibition by cyanate and carbon dioxide are consistent with a I:i molar stoichiometry of this binding.
Benesch 3 Benesch and Enoki (13) suggested
that the site of binding might be on the diad axis of syr~netry between the beta chains.
Perutz (14) proposed 3 on the basis of model fitting and the
data of Bunn and Briehl (2) 3 that two of the paired charged groups involved were the ~ amino groups of the N-terminal valine residues in the beta chains. Kilmartin and Rossi-Bernardi (8) have shown that carbon dioxide binds these terminal amino groups.
Moreover 3 the competition of carbon dioxide and
2~3-DPG for these sites has been inferred from the interference of carbon dioxide with the 2~3-DPG effect on the oxygen equilibrium of hemoglobi% as shown recently by Tomita and Riggs (15). The inhibition of 2~3-DPG
1508
Vol. 44, No. 6, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
binding to hemoglobin, per se, by both carbon dioxide and cyanate provides strong evidence that the ~ amino groups participate in the binding site of hemoglobin for 2,3-DPG. ACKNOWLEDGEMENT This work was supported by USPHS Grants # AM-13430, AM-13698, AM-54353 AM-15053, HE-02001, HE-05741, HE-05443 and a grant-in aid from the New York Heart Association.
The authors are Career Scientists of the Health Research
Council of the City of New York.
REFERENCES I. 2. 3. 4. 5. 6o 7. 8. 9. i0. ii. 12. 13.
14. 15.
Benesch, R., R.E. Benesch and C.I. Yu, Proc. Nat. Acad. Sci. 59, 526 (1968). Bunn, H.F. and R.W. Briehl, J. Clin. Invest. 49, 1088 (1970). Stark, G.R. and D.G. Smyth, J. Biol. Chem. 238, 214 (1963). Kilmartin, JoV. and L. Rossi-Bernardi~ Nature. 222, 1243 (1969). Cerami, A. and J.M. Manning, Proe. Nat. Acad. Sci. 683 1180 (1971). Drabkin, D.L., J. Biol. Chem. 164. 703 (1946). Craig~ L.C. Advan. Anal. Chem. Instr. 41, 35 (1965). Kilmartin, J.V. and L. Rossi-Bernardi, NASA SP-188, 73 (1969). Scatchard, G., Ann. N.Y. Acad. Sci. 51, 660 (1949). Beneschj R.E., R. Benesch and C.I. Yu~ Biochem. 8, 2567 (1969). Rose, Z.B., Fed. Proceed. 29, 1105 (1970). Minakami, S. and H. Yoshikawa, J. Biochem. 59, 145 (1966). Benesch, R., R.E. Benesch and Y. Enoki, Proc. Nat. Aead. Sci. 61, 1102 (1968) o Perutz, M.F., Nature 228, 734 (1970). Tomita, S. and A. Rigg% J. Biol. Chem. 246, 547 (1971)o
1509