The Mechanism of Interaction of Red Cell Organic Phosphates with Hemoglobin

THE MECHANISM OF INTERACTION OF RED CELL ORGANIC PHOSPHATES WITH HEMOGLOBIN By RUTH E. BENESCH and REINHOLD BENESCH Department of Biochemistry, Columbia University, College of Physicians 6.Surgeons, N e w York, N e w York

I. General Introduction . . . . . . . . 11. Interaction of Organic Phosphates with Hemoglobin . . A. Organic Phosphates of the Red Cell . . . . B. Effect of DPC on the Oxygenation Curve of Hemoglobin C. Location of the DPG Binding Site . . . . . . . . . . . . 111. DPC and the Bohr Effect IV. DPC and C02 Transport . . . . . . . . V. Effect of DPC on Abnormal Hemoglobins . . . . . VI. Organic Phosphates and Artificial Hemoglobin Hybrids . . . . . . . VII. Summary and Conclusions . References . . . . . . . . . .

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211 216 216 216 221 227 230 230 232 233 234

I. GENERALINTRODUCXION Very little of the oxygen required by higher organisms reaches the tissues by diffusion alone. As much as 9% is carried by hemoglobin from the lungs to the periphery. Loading of hemoglobin with oxygen in the lungs is essentially complete under normal conditions, since the alveolar oxygen pressure is more than enough to saturate the protein (Fig. 1). pop vm.

pOz art.

FIG. 1. Oxygen equilibrium curve of hemoglobin in whole blood. At a venous oxygen pressure of 40 mm, 38%of the oxygen is unloaded. Because of the steepness of the curve in this region, a drop in venous pol to 30 mm would result in the release of an additional 18%of the oxygen. ven., venous; art., arterial. 211

212

RUTH E. BENESCH AND REINHOLD BENESCH

Oxygen release, on the other hand, depends crucially both on the shape of the oxygen binding curve and on the overall oxygen affinity. The steep middle portion of the sigmoid oxygenation curve (Fig. 1) makes oxygen release particularly sensitive to small changes in oxygen pressure in the physiological range. As a result, the oxygen carrier can respond readily to changes in oxygen requirement, for example during exercise, without substantial changes in tissue oxygen tension, A control mechanism which shifts the steep portion of the oxygenation curve into the required range of oxygen tensions in response to physioIogicaI and pathological changes in tissue oxygenation is therefore clearly required for efficient oxygen transport. The familiar Bohr effect does, in fact, change the oxygen affinity of hemoglobin as a function of hydrogen ion concentration. As a result of this reciprocal affinity of hemoglobin for oxygen and hydrogen ions, oxygen release is facilitated in the periphery as carbonic acid accumulates in the red cell (Fig. 2). The molecular basis of the Bohr effect has long been recognized as a change in acid strength of some amino acid residues when the conformation of the protein changes with ligand binding (Wyman, 1948). Recent work has led to the identification of some of the groups involved (Perutz et al., 1989; Kilmartin and Rossi-Bernardi, 1969). It has been known for over fifty years that the oxygen affinity of blood increases when the red cell contents are diluted by hemolysis. This led Barcroft to speculate as early as 1921 whether there was “some third substance present :. , which forms an integral part of the oxygen-hemo-

0

20

40

60 80 DO* (rnhHg)

100

120

140

FIG. 2. Effect of pH on the oxygenation curve of hemoglobin (Bohr effect).

ORGANIC PHOSPHATES AND HEMOGLOBINS

213

globin complex?” (Adair et al., 1921). It is, therefore, surprising that Greenwald’s discovery of high concentrations of ~-2,3-diphosphoglycerate (DPG)l in porcine red cells in 1925 (Greenwald, 1925), and the subsequent demonstration that this holds for many other species including man (Rapoport and Guest, 1941), did not prompt an investigation of its effect on oxygen transport until recently. A series of investigations by Chanutin and his co-workers demonstrated that DPG can form complexes with hemoglobin in solutions of low ionic strength (Chanutin and Curnish, 1965a,b), but it was not until February, 1966, that the dramatic effect of this compound in lowering the oxygen afsnity of hemoglobin was finally described (Benesch and Benesch, 1967a,b). It was confirmed in July of the same year by Chanutin and Curnish (1967), and since then a voluminous literature has accumulated on the subject. As a result, some new insights in physiology and medicine have been brought to light. These will be discussed only briefly in the course of this article, and the reader is referred to a number of recent reviews (Brewer, 1970; Rgrth and Astrup, 1972; deVerdier et al., 1969; Benesch and Benesch, 1969,1970; Bunn and Jandl, 1970; Klocke, 1972). As will be discusged in more detail below, the affinity of hemoglobin for oxygen and organic phosphates is reciprocal, analogous to the Bohr effect. The oxygen affinity of hemoglobin should therefore vary inversely with the DPG concentration. Thus, it is almost certain that DPG, since it is diluted on hemolysis, was Barcroft’s “third substance” and is responsible for the lower oxygen affinity of hemoglobin in the intact red cell. The predicted relation between DPG concentration and oxygen affinity inside the erythrocyte holds true in a number of diverse conditions, both in uiuo and in uitro. Thus, the drop in intracellular DPG which occurs in experimental acidosis (Rapoport, 1936) and also during blood storage (Bartlett and Barnet, 1960) is accompanied by a corresponding increase in oxygen affinity (Bunn et al., 1969). The parallel drop in p5,,and DPG concentration in stored blood is illustrated in Fig. 3. After transfusion of DPG-depleted blood, the oxygen affinity of the recipient’s blood is increased (Valtis and Kennedy, 1954), but eventually it returns to normal owing to the & nouo synthesis of DPG within the host ( Valeri and Hirsch, 1969). An example of a change in the opposite direction is the rapid and substantial increase in DPG levels which is seen on ascent to high altitudes. The elevation of the DPG level is well correlated with the decrease in the oxygen affinity of the blood at these Abbreviations used are: DPG, 2,3-diphosphogIyceric acid; ATP, adenosine triphosphate; PLP, pyridoxal 5’-phosphate; IHP, inositol hexaphosphate; bis-Tris, bis( Zhydroxyethyl ) iminotris ( hydroxymethy1)methane; Tris, tris( hydroxymethyl) aminomethane; NMR, nuclear magnetic resonance.

214

RUTH E. BENESCII AND REINHOLD BENESCH

Days Storage ( 4 O C )

Days Storage (4'C)

FIG.3. Changes in p~ and 2,3-diphosphoglyceric acid ( 2,3-DPG) concentration during storage of blood in acid citrate dextrose (ACD). Open and filled symbols represent blood samples from two different donors; pso is the oxygen pressure for 50% oxygenation ( Dawson, 1970).

altitudes (Fig. 4). Both changes are reversed on return to normal barometric pressure. It is noteworthy that the DPG response to hypobaric oxygen pressure occurs only in subjects who exercise (R@rthet al., 1972). The rapidity of the response suggests that the DPG mechanism is a major factor in the short-term adaptation of man to high altitudes. Some very clear-cut examples of the interrelationship between red cell DPG levels and oxygen delivery come from .the study of certain congenital red cell enzyme deficiencies. Thus, in pyruvate kinase deficiency, where impaired breakdown leads to high DPG levels, the oxygen affinity sinks to very low values (Mourdjinis et al., 1969; Delivoria-Papadopoulos et al., 1969) (Table I ) . In the case of two siblings with this anemia, Mourdjinis et al. (1969) estimated that the decrease in oxygen affinity would permit the release of as much oxygen from 9 g of hemoglobin per 100 ml of blood as from normal blood containing 15 g of hemoglobin per 100 ml. By contrast, the decreased synthesis of DPG associated with hexokinase deficiency is accompanied by an increase in the affinity of the carrier for oxygen ( Delivoria-Papadopoulos et al., 1969). In this metabolic defect the delivery of oxygen is therefore markedly impaired, and its clinical consequences are also much more severe than those of pyruvate kinase deficiency. In general there is now a large body of evidence that in a variety of diseases associated with hypoxemia, such as low cardiac output, lung disease and anemias, the red cell responds with an increase in the DPG level and a lowered affinity of the hemoglobin for oxygen (Oski et al., 1969; Lenfant et al., 1970; Edwards et al., 1968; Valeri and Fortier, 1970).

215

ORGANIC PHOSPHATES AND HEMOGLOBINS

264 24

I

I

I

I

0

24

40

1

I

72

96

I

1

I

120 144/0

I

I

24

40

72

Hours

FIG.4. Effect of exposure to high altitude on blood oxygen affinity and 2,3-diphosphoglycerate ( DPG ) concentration. Open circles, pw, filled circles, DPG concentration, From Lenfant et al. (1968).

In the case of sickle cell anemia, the elevation of intracellular DPG is a mixed blessing, since the stabilization of deoxyhemoglobin S by DPG will favor sickling. DPG synthesis in the red cell results from a shunt in the glycoIytic pathway (Rapoport and Luebering, 1950, 1951, 1952) and is subject to multiple feedback control (Rapoport and Luebering, 1952; Dische, 1941; Joyce and Grisolia, 1959; Rose, 1968). We are thus dealing with an adaptive mechanism which acts to increase oxygen delivery to the tissues when less oxygen is available. TABLE I Oxygen Aj'inity and DPG Concentration in Pymvate Kinuae and

Hexokinase Deficieny

Subject

DPG (pmoles/lOO ml RBC)

Normal Normal Pyruvate kinase deficiency Pyruvate kinase deficiency Hexokinase deficiency

340 4005 408 f 52b 664" 1027* 274b

Mourdjinis et al. (1969). Delivoria-Papadopoulos et al. (1969).

-

P60

26.2 f 0.6O 2 4 . 4 f 0.9 3 8 . 2 0.15 38' 19

*

216

RUTH E. BENESCH AND REINHOLD BENESCH

11. I N T E R A ~ O OFNORGANIC PHOSPHATESWITH HEMOGLOBIN In order to facilitate the subsequent discussion, the term “helcotropic” is introduced to characterize compounds which alter the affinity of hemoglobin for ligands (Greek d&s-attraction; T w e t u r n , change) .* A. Organic Phosphates of the Red Cell Among the organic phosphates of the red cell which lower the oxygen afEnity of hemoglobin, DPG is the most abundant. Its molar concentration in human erythrocytes is nearly equal to that of hemoglobin, i.e., about 5 mM (Rapoport and Guest, 1941). The only other intracellular phosphate ester present in sufficient concentration (about 1.3 mM) to contribute significantly to modifying the oxygen affinity of hemoglobin is adenosine triphosphate (ATP). However, as pointed out by Bunn et al. (1971), its high affinity for the intracellular magnesium ion largely eliminates ATP as a helcotropic factor. The red cell inorganic phosphate concentration is only about 0.5 mM. DPG is not the only major helcotropic phosphate of erythrocytes. In some species, such as birds and turtles, it is replaced by inositol hexaphosphate (Rapoport and Guest, 1941) or, according to a recent claim (Johnson and Tate, 1%9), inositol pentaphosphate. Some species, such as the ungulates and the cat, lack significant amounts of helcotropic phosphates altogether (Rapoport and Guest, 1941). B. Effect of DPG on the Oxygenation Curve of Hemoglobin It is now clear that many of the data on ligand binding before 1966 were obtained on hemoglobin containing variable and unknown amounts of phosphate cofactors. Dialysis against distilled water, which was commonly used as the final step in hemoglobin purification, is ineffective for separating these compounds from hemoglobin ( Benesch et at., 1968a). For the complete removal of these highly charged molecules it is necessary to provide an ionic strength of at least 0.1 and a pH above the isoelectric point of hemoglobin, i.e., higher than 7. Under these conditions both dialysis and gel filtrationa have consistently been reported to ‘We are indebted to Dr. John Karkas for his advice in the choice of this term. ‘At p H values below neutrality significant amounts of DPG remain bound to hemoglobin, and this presumably accounts for the reported failure in one laboratory to strip hemoglobin adequately (Diederich et d.,1969). It should also be noted that in neutral solution the separation between DPG and hemoglobin in gel filtration is smaller than expected from its molecular weight. This is due to repulsion between the negative charges, which gives the molecule an extended conformation and increases its mobility to that of a compound of about four times its molecular weight ( Berman et al., 1971).

ORGANIC PHOSPHATES AND HEMOGLOBINS

-1.0

0

log

por

+LO

217

+2.0

FIG. 5. Comparison of the oxygenation curve of myoglobin, “stripped” hemomyoglobin at 30°C plotted from the data of globin and whole blood. -0 Rossi-Fanelli and Antonini (1958); 0-0 “stripped hemoglobin in 0.01 M NaCl at 30°C, pH 7.0 (before deoxygenation) (Benesch and Benesch, 1969); whole blood at 30°C plotted from the data of Astrup et al. ( 1965),

(>--a,

yield hemoglobin containing 0.03 mole of DPG per mole or less (Benesch et al., 1968a; Berman et al., 1971; Bunn and Briehl, 1970; Ogata and McConnell, 1971; Lindstrom and Ho, 1972; Tyuma and Shimizu, 1969; Gibson, 1970). A study of the equilibrium between oxygen and hemoglobin devoid of cofactors thus became feasible, and this is illustrated in Fig. 5. The most striking property of such “stripped hemoglobin is its very high oxygen affinity. In solutions of low ionic strength it actually falls into the range of that of myoglobin (Fig. 5 ) . The familiar assumption that hemoglobin has a much lower affinity for oxygen than myoglobin is therefore true only in the presence of helcotropic cofactors. The effect of DPG on ligand binding by hemoglobin is now firmly established (R. Benesch et al., 1968a; R. E. Benesch et al., 1969a, 1971b; Bunn and Briehl, 1970; Ogata and McConnell, 1971; Gibson, 1970) and is illustrated in Fig. 6. These measurements were made in solutions of approximately physiological p H and ionic strength, Le., 0.1 M C1- and pH 7.3. Bis-Tris was chosen as a buffer, since, owing to its low pK ( 6 . 5 ) , 80%of it is in the uncharged form at p H 7.3 and because it had no influence on the oxygenation curve of .hemogIobin over a 25-fold concentration range. The increasing resistance of hemoglobin to oxygenation with rising DPG concentration (Fig. 6 ) clearly points to a stabilization of the deoxy as against the oxy conformation by the organic phosphate. Direct measurements of complex formation between DPG and hemoglobin show

218

R U T H E. BENESCH AND REINHOLD BENESCH

log P O 2 FIE. 6. Effect of 2,3-diphosphoglyceric acid (DPG) on the oxygen equilibrium curve of hemoglobin (Hb). Hb concentration, 6 x lo-' M; bis (2-hydroxyethy1)iminotris(hydroxymethy1)methane buffer (pH 7.3), 0.05 M; total chloride, 0.1 M ; temperature, 2OOC. X-x, NO DPG; 0-0, 1.1 X 10-'M DPG; A-A, 2.5 X 10-4 M DPG; 4.0 x 10-~M DPG; v--0, 6.0 x M DPG; .---a, 10.0 x loe4A4 DPG; A-A, 25.0 x lO-'M DPG. From Benesch et al. (1971b).

0-a,

that under conditions identical with t h s e used for the oxygen binding cumes in Fig. 6, a single molecule of DPG is bound per mole of deoxyhemoglobin, but not by oxyhemoglobin (Fig, 7 ) . The half-saturation point of the binding curve in Fig. 7 corresponds to an association constant of 6.7 X lo4 M-* or a free energy of binding of about -6.5 kcal/mole. Similar conclusions were reached by Lo and Schimmel ( 1969), Pace et al. ( 1970), Ogata and McConnell ( 1971), and De Bruin and Janssen (1973). Arnone ( 1972) has recently provided a most satisfying structural basis for the stoichiometry and specificity of DPG binding to deoxyhemoglobin. Binding of organic phosphates to hemoglobin can occur in many nonspecific ways, depending on such factors as ionic strength and pH. This accounts for reports that DPG and related compounds are also bound significantly to oxyhemoglobin ( Chanutin and Hermann, 1969; Garby et al., 1969). Hemoglobin is, after all, a polyelectrolyte with many POtential binding sites for a polyanion like DPG. However, it cannot be too strongly emphasized that the only binding site which matters from a functional point of view is the oxygenation-linked one ( Wyman, 1948). This is completely analogous to the Bohr effect, where, among the many protons bound by both oxy- and deoxyhemoglobin, only the ones whose affinity is different for oxy- and deoxyhemoglobin are relevant for the shift in oxygen affinity with pH.

219

ORGANIC PHOSPHATES AND HEMOGLOBIN'S

Free (DPG) x lo5 FIG.7. Binding of 2,3-diphosphoglyceric acid (DPG) by hemoglobin ( Hb) Hb concentration, 1 x lo-' M ;bis( 2-hydroxyethyl)iminotris(hydroxymethy1)methanebuffer, pH 7.3, 0.05 M; total chloride, 0.1 M ;temperature, 20°C. 0,deoxyhemoglobin; 0 , oxyhemoglobin. The points are experimental and the line is calculated for a M . From Benesch et al. single site with a dissociation constant of 1.5 X (1971b).

.

In the case of DPG, the oxygenation linked binding is also the strongest one, so that in relatively dilute solution, and physiological conditions of pH and ionic strength, only this interaction is observed. The treatment used originally by Wyman (1948) for the quantitative formulation of the Bohr effect is equally applicable in this case, so that the shift in the oxygenation curve as a function of DPG concentration can be predicted from the experimentally observed binding constant of DPG to deoxyhemoglobin. Figure 8 shows the relation between p50 and DPG concentration. The points are taken from Fig. 6, and the curve is calculated from Eq. ( l } (Renthal, 1972).

I

I

-4.0

1

-3.5

1

-3.0

I

-2.5

log [DPGJ FIG.8. Relation between pa0 and 2,3-diphosphoglyceric acid (DPG) Concentration. The points are experimental (Fig. 6), and the line was calculated from Eq. (1) with KD = 1.5 X lO-'M and R = 3.0 X lO-'M. From Benesch et d. (1971b).

220

RUTH E. BENESCH AND REINHOLD BENESCH

where p50 is the oxygen pressure at 50%oxygenation, n is Hill’s coefficient and K, is the measured dissociation constant of the deoxyhemoglobinDPG complex, i.e., 1.5 X M at 20°C. K’ is a fitting parameter, which, since fully oxygenated hemoglobin does not bind DPG under these conditions, must represent binding to intermediate states of oxygenation, such as HbO, and HbO,. It is clear that the magnitude of the observed shift in the oxygenation curve induced by DPG over a 30-fold concentration range is quantitatively accounted for by its association with deoxyhemoglobin when it is assumed that its affinity for partially oxygenated states is at least 20 times lower ( Benesch et al., 1971b). An increasing body of evidence now indicates that the DPG bound to deoxyhemoglobin is released with the binding of the second or third oxygen when the change in quaternary structure from the deoxy to the oxy conformation takes place. Thus, MacQuarrie and Gibson ( 1971) have measured the release of the cofactor and ligand binding simultaneously, using pyrene trisulfonate, a fluorescent analog of DPG, and found that the compound was set free with the binding of the third ligand. Tyuma and collaborators (1971, 1972,1973) reached a similar conclusion from oxygen equilibrium studies. They calculated the binding constants of the four oxygenation steps in the presence and in the absence of DPG on the basis of the Adair model (Table 11). It can be seen that only the a n i t y of the last oxygen (k,) is not lowered by DPG. The values of k, and k, are decreased about equally, but the largest effect is associated with k,. The correlation between the gain of 2 to 3 ligand molecules and the loss of the organic phosphate has also been proposed by Perutz (1970), Shulman et al. (1971), Herzfeld and Stanley (1972), Ogata and McConnell ( 1972b), and Huestis and Raftery ( 1972). TABLE I1 Effect of DPD ma Ihe Intrinsic Microscopic Associulim Constants of Oxygen for Hemoglabin A4

Hemoglobin “Stripped” “Stripped” plus 2 X 10-8 M DPG

R1

k2

kr

kc

0.024 0.010

0.074 0.023

0.086 0.008

7.4 11.2

a The constants were calculated by application of the Adair equation to the results of measurements on 1.5 x M Hb solutions in 0.05 M bis-Tris buffer pH 7.4, 0.1 M NaCl at 25°C (Tyuma et al., 1973; for further data consult Table I11 in that article).

221

ORGANIC PHOSPHATES AND HEMOGLOBINS

The actual mechanism of DPG release depends on the model which one assumes for the oxygenation of hemoglobin. On the basis of the two-state model of Monod et al. (1965), DPG will be released as the ratio of T to R conformation changes with oxygenation since the cofactor is only bound by the T state. The multifunctional attachment of DPG to deoxyhemoglobin by a number of salt bridges (see below) leaves open the possibility, however, that these are broken sequentially with the uptake of successive oxygen molecules as expected from the “induced fit” model of Koshland et al. (1966). C . Location of the DPG Binding Site

The choice of a binding site was facilitated by the simple 1:1 stoichiometry of the interaction. It is now clear that only tetramers in the deoxy conformation have a site with a high affinity for a single molecule of the cofactor. This applies not only to deoxy as against liganded hemoglobin A, but also to the p4 tetramer. It has a conformation close to that of deoxyhemoglobin and maintains it even in the presence of oxygen (Perutz and Mazzarella, 1963). It also binds 1 mole of DPG per mole of tetramer with an affinity which is unchanged by oxygenation (Benesch et al., 1968b). Furthermore, the oxygen affinity of p4 hemoglobin is uninfluenced by DPG, in excellent agreement with the concept that the molecular basis for the helcotropic effect is a differential &nity for two different conformations. Significantly, when p4 tetramers are dissociated by mercuration, DPG binding is lost. Similarly, chains, which are essentially monomeric, entirely fail to complex DPG (Benesch et d.,1968b). The minimum qualifications for a suitable binding site were, thus, that it be symmetrical to accommodate the single effector molecule per tetramer, that its geometrical dimensions change drastically from deoxy to oxyhemoglobin, and that it contain a sufficient number of positively charged groups for binding the polyanion. Perutz et ul. (1968) and Benesch et al. (1968b) recognized that the entrance to the central cavity, on the diad axis, fulfills these requirements. Our finding that isolated p, but not 0 , subunits combine with DPG supports this conclusion, since the site in question is formed by the N and C terminal ends of the two p chains. The electrostatic nature of the interaction between the polyanion and the protein precludes the isolation and identification of a DPG-containing fragment. One solution to this problem became available with our discovery (Benesch et al., 1969b, lWla, 1972; Renthal, 1972) that pyridoxal phosphate (PLP) can be used as an affinity label since it has analogous helcotropic properties to DPG and competes with it for the same site in (Y

222

RUTH E. BENESCH AND REINHOLD BENESCH

deo~yhemoglobin.~At the same time, it can be attached covalently to the protein, since the initially formed Schiffs base is reduced by sodium borohydride to a stable secondary amine (Fischer et al., 1958). A search for the fluorescent label after enzymatic hydrolysis revealed that over 90%was attached to the N-terminal valine residue of the ,8 chain. The attachment of the label to this site is highly specific for deoxyhemoglobin. However, unlike DPG, PLP also reacts with liganded hemoglobin, but at a different site, i.e., the N-terminal residue of the chain. The high reactivity of PLP with terminal NH, groups in hemoglobin is altogether remarkable since in all enzymes for which PLP is a cofactor it is invariably found on the r-amino groups of lysine ( Fasella, 1967). Hemoglobin with only one covalently attached PLP molecule can only be formed and isolated under strictly anaerobic conditions. This molecule, i.e., a2pPpLpis stable only in the deoxy conformation which does not significantly dissociate into a/? dimers (Benesch et al., 1962, 1964; Kellett, 1971). A recent estimate by Thomas and Edelstein (1972) of the dissociation constant of the deoxy tetramer is 3 X 10-l2M compared to 2 X M for the liganded form. On admission of oxygen, therefore, a rearrangement involving subunit exchange takes place (Guidotti et al., 1963) as shown in the following scheme: (Y

2azflflpLY

F? 2 4 3

lt

azflz

+

2orfiPLP

t

az&PLp)z

Hemoglobin containing 2 PLPs per tetramer which is formed by this exchange has been isolated in the expected yield by chromatography on phosphocelldose.. This molecule has a permanently lowered oxygen affinity (Fig. 9), which is not further affected by addition of DPG. Very recently, a definitive description of the DPG deoxyhemoglobin complex has appeared (Arnone, 1972). Crystals of deoxyhemoglobin were soaked in a 70!%solution of 2-methyl-2,4-pentanedioIwith and without 1 mM DPG for the determination of difference electron density maps. This ingenious procedure made it possible to maintain electrostatic cohesion between the cofactor and the macromolecule in spite of the high salt concentration necessary for growing adequate crystals. The crystallographic evidence confirms the binding of a single molecule and defines the amino acid residues which participate in binding it (Fig, 10). The N-terminal valines, the histidines 2 and the histidines 143 are involved in electrostatic interaction with the phosphate groups, while the lysines

' Other pyridoxal derivatives which lack either the Schiffs base-forming aldehyde group, or the phosphate side chain, or both, are totally inactive. Moreover, while the DPG effect is blocked by PLP, a normal DPG response occurs in the presence of all the inactive pyridoxal derivatives.

ORGANIC PHOSPHATES AND HEMOGLOBINS

223

Log PO2

FIG. 9. Oxygenation curves of pyridoxylated hemoglobin. Hemoglobin concen-

tration 5 x lo-‘ M in 0.05 M bis( 2-hydroxyethyl)iminotris( hydroxymethy1)methane (pH 7.3), 0.1 M C1-, 20°C. A, “stripped” hemoglobin; A, “stripped” hemoglobin PIUS 2.5 X lo-’ M DPG; 0, Hb( PLP),; 0, Hb( PLP), P ~ U 2.5 S X lo4 M DPG.

FIG. 10. Sketch showing the binding of 2,3-diphosphoglyceric acid (DPG) to human deoxyhemoglobin. The stereochemistry of DPG complements the basic residues of the central cavity to form salt bridges with valines 1and histidines 2 and 143 of both p-chains, and with lysine 82 of one &chain. This binding pulls the A-helix and the hemoglobin S mutation site (residue 6) toward the E-helix and ,the EF corner. From h o n e ( 1972).

224

RUTH E. BkNESCH AND REINHOLD BENESCH

82 share the binding of the carboxyl group. The symmetry of this site is in agreement with our earlier observation (Benesch et al., 1969a) that the L-isomer of DPG has identical helcotropic effects to the naturally oc cu r ~ n gn-isomer. It is highly significant that in the presence of the cofactor the site is modified, principally by a movement of the A helices. In this way a close fit is achieved leading to the substantial stabilization of the deoxy conformation by 6.5 kcal, derived from binding and oxygenation measurements in solution ( Benesch et al., 1969a, 1971b). The constellation of positive residues in the stereochemical arrangement present in the deoxyhemoglobin tetramer provides a unique and specific binding site for polyphosphate cofactors. Conversion to the oxy conformation distorts the site sdciently to expel DPG completely. However, even substitution or elimination of one of the residues involved leads to a drastic decrease in the interaction energy. For example, HbAIc, in which p N l is blocked with a hexose, has only a slight affinity for DPG (Bunn and Briehl, 1970), and both HbF, (Bunn and Briehl, 1970) and cat B hemoglobin (Taketa et al., 1971), in which this group is acetylated, fail to respond to DPG. Similarly, the minimal effect of DPG on sheep hemoglobin ( Bunn, 1971) can be ascribed to the deletion of p N2 His in this species (Perutz, 1970). The other histidine of the binding site, i.e., p-143, is replaced by a serine in fetal hemoglobin and here, too, the effect of the cofactor is drastically weakened in comparison with adult hemoglobin (Tyuma and Shimizu, 1969; Bauer et al., 1968).5 While the interaction between the helcotropic cofactor and hemoglobin therefore depends on a rigidly defined protein site, it has become clear that this site can be saturated by a wide variety of small molecules. Thus, e.g., ATP, like DPG, reacts with hemoglobin in a 1:l molar ratio and with a comparable affinity (Lo and Schimmel, 1969). Its helcotropic effect is, in fact, identical at 37"C, although it has a smaller temperature coefficient( Benesch and Benesch, 1970; Bunn et d.,1971). A spin-labeled ATP and a spin-labeled triphosphate were used as conformational probes by Ogata and McConnell ( 1971, 1972a). Both these compounds react with deoxyhemoglobin, but not at all with OXYhemoglobin, with a binding stoichiometry of 1 molecule per hemoglobin Fetal blood has a higher affinity for oxygen than maternal blood ( Haselhorst and Stromberger, 1930; Eastman et al., 1933), and this is assumed to facilitate oxygen transport across the placenta. However, neither the oxygen affinity of purified HbA and HbF nor the DPG concentration of fetal and maternal red cells differ significantly. Therefore, the discovery that the helcotropic effect of DPG on HbF is weaker because of its defective binding site, provides a molecular explanation for this phenomenon.

ORGANIC PHOSPHATES AND HEMOGLOBINS

225

tetramer as measured both by equilibrium dialysis and by paramagnetic resonance. The observed affinity constants were comparable to that of DPG under similar conditions. The expected helcotropic effect was demonstrated by measurements of the oxygen equilibria. As mentioned earlier, PLP is another example of a polyfunctional organic phosphate which can fully substitute for DPG. Even pyrene trisulfonate without any phosphate groups has been used as a fluorescent analog of DPG, (MacQuarrie and Gibson, 1971) although its affinity for the deoxy site is much weaker, and it also shows significant binding to carbonmonoxyhemoglobin. Among the phosphate esters investigated inositol hexaphosphate, the principle organic phosphate of the avian red cell, shows by far the largest helcotropic effect (Benesch et al., 1968a). This compound has eight negative charges at neutral pH, where DPG has only between three and four. It is therefore, able to neutralize more fully the four pairs of positive residues in the site, and this leads to very tight complex formation. On the other hand, it is significant that the phosphonic acid analog of 2,3-DPG decreases the oxygen affinity of hemoglobin only very little (Benesch et al., 1973). Both the smaller size of this molecule and the reduced number of negative charges undoubtedly affect adversely its fit to the binding site. Among the phosphates, monophosphoglycerates are much less active than DPG and inorganic phosphate is even less effective. Nevertheless, it is of fundamental importance that the helcotropic effect of DPG can be interpreted as a limiting case of the well known effect of salt on the ligand affinity of hemoglobin (Rossi-Fanelli et al., 1961; Enoki and Tyuma, 1964; Barcroft and Roberts, 1909; Barcroft and Camis, 1909; Benesch et al., 1969a). Thus we have shown that the DPG effect disappears in the presence of 0.5 M NaCl ( Fig. ll,C, p. 226), and conversely that saturation of hemoglobin with DPG eliminates the influence of neutral salt (Fig. 11) (Benesch et al., 1969a). However, for the same shift in oxygen affinity, the NaCl concenfration must be about 104 higher than that of DPG. This illustrates the effectiveness of the intracellular cofactor with the properties of a “supersalt” (Benesch and Benesch, 1970) which can regulate the oxygen affinity within physiologically required limits without damage to the osmotic health of the red cell. Ionic strength is therefore of crucial importance in assessing the magnitude of helcotropic effects (Fig. 11). In low salt concentration the shift is so large that in the presence of less than 1 mole of cofactor per tetramer two separate steps in the oxygenation curve can be distinguished which correspond to the successive oxygenation of the phosphatefree and phosphate complexed fractions. This is particularly clear when

226

RUTH E. BENESCH AND REINHOLD BENESCH

,/// 20 -0.4 - 0 2

0

02

04

06

08

10

1.2

log poe

FIG. 1 Influence of ionic strength on the helcotropic effect of 2,3-di~--xphcglyceric acid (DPG). ( A ) 0.01 M C1-; ( B ) 0.10 M C1-; ( C ) 0.50 M C1-. Hemoglobin concentration 6 x 10“ M in 0.05 M bis( 2-hydroxyethyl)iminotris(hydroxymethyl)methane, pH 7.30, temperature 20°C. 0,“Stripped” hemoglobin; 0 , “stripped” hemoglobin plus 2.5 X 10“ M DF’G. Data from Benesch et d. (1969a).

a cofactor with a very high affinity, such as inositol hexaphosphate (IHP), is employed. When fractional amounts of this compound are used, sharply biphasic oxygenation curves are obtained (Fig. 12) which show the expected proportion of high and low affinity hemoglobin. Furthermore, it should be noted that even l mole of cofactor per mole of tetramer gives rise to a flattening in the oxygenation curve, since the free organic phosphate concentration rises as oxygenation proceeds. A curve with a slope comparable to that of stripped hemoglobin is obtained only when the concentration of organic phosphate is sufficiently high to maintain it at a constant level throughout the oxygenation (Benesch et al., 1968a; Ogata and McConnell, 1971; Herzfeld and Stanley, 1972; Tomita and Riggs, 1971). The steepness of the oxygenation curve of whole blood, where the total DPG concentration is similar to that of hemoglobin is, therefore, puzzling. The affinity of inositol hexaphosphate for deoxyhemoglobin has been estimated to be at least loRM-l (Gray and Gibson, 1971). It has, therefore, been used extensively for maintaining a variety of hemoglobins in

ORGANIC PHOSPHATES AND HEMOGLOBINS

/x-

227

J

log P O 2

FIG. 12. Effect of inositol hexaphosphate (IHP) on the oxygenation of hemoglobin. Hemoglobin concentration; 0.3% (4.6 X lo6 M ) in 0.01 M NaCl, pH 7.0 (before deoxygenation), temperature 10°C. X, No IHP; A, 1.2 X lo-' M IHP (0.26 mole/mole); 0, 2.3 X 1O-'M IHP (0.5 mole/mole); 0, 3.5 X 104M IHP (0.76 mole/mole); A,4.6 X M IHP ( 1.0 mole/mole). From Benesch et ol. (1968a).

the deoxy conformation ( T state) in experiments designed to probe the conformational changes which accompany ligand binding. It was particularly effective for the recognition of the relative contributions of (Y and /3 subunits in the reaction with ligands (Olson and Gibson, 1970, 1971, 1972a; Lindstrom et al., 1971; Cassoly et al., 1971; Lindstrom and Ho, 1972; Ogawa and Shulman, 1971; Shulman et at., 1971). A recent example of the powerful effect of IHP in stabilizing the T state comes from the studies of Bonaventura et al. (1972) on carboxypeptidase A-digested hemoglobin. This hemoglobin derivative, which lacks the two C-terminal residues of the /3 chains, has a very high oxygen affinity and no heme-heme interaction, and crystals in the oxygenated and deoxygenated state are isomorphous. Addition of excess IHP causes a 5-fold decrease in the oxygen affinity, and oxygenation becomes cooperative; this is consistent with the idea that IHP displaces the equilibrium between the R and T state in favor of the latter.

111. DPG

AND THE

BOHREFFECX

The relation between these two major helcotropic mechanisms is of great interest, but a number of studies on the influence of DPG on the Bohr effect have led to conflicting results. However, an analysis of the existing data makes it clear that the disagreement is only apparent and is due to an unwarranted assumption. As already pointed out by Wyman in 1964, the simple form of the linkage differential equation, Eq. ( 2 )

228

RUTH E. BENESCH AND REINHOLD BENESCH

is valid in the presence of a third component only if its activity is not affected by ligand binding. This condition obviously does not apply to DPG with its enormous differential affinity for the oxy and deoxy conformations. The situation is further complicated by the dependence of DPG binding on hydrogen ion concentration, Therefore, the Bohr effect as measured by the change in p50 with pH (Bohr coefficient), on the one hand, and by the number of hydrogen ions released on oxygenation at constant pH (Haldane coefficient) on the other, will not be equal:

(slog

pso) /ApH

# An+[+.

Wyman even predicted that in such a case the extent of the inequality will be a measure of the differential affinity of the third component for the liganded and ligand-free form of the protein. It is, therefore, not surprising that the reports in the literature fall into two categories, depending on which one of these coefficientswas used to measure the influence of DPG on the “Bohr effect.” Differential titration has shown (De Bruin et al., 1971; Bailey et aZ., 1970) that DPG increases the number of protons exchanged on ligand binding above pH 7 and decreases it below this value. This result bears out Arnone’s assumption (Arnone, 1972) that the binding of the polyanion is accompanied by an increase in pK of the positive groups on the protein and a decrease in pK of its own phosphate groups. A titration curve of DPG (Kiessling, 1934; Benesch et al., 1969a) is shown in Fig. 13. The

FIG. 13. Titration curve of 2,3-diphosphogly~rate. 5 X M diphosphoglyceric acid in 0.1 N NaCl was titrated with 0.5 N NaOH. From Benesch et d.

(196%).

229

ORGANIC PHOSPHATES AND HEMOGLOBINS

first buffer region presumably involves one proton from each phosphate and that of the carboxyl group. The remaining protons ionize in the neutral range and their pK must be lowered by interaction with the protein. As expected frorh the binding of a single mole of DPG, the differential proton absorption reaches a limiting value at a DPG:Hb ratio between one and two. In the presence of DPG the proton exchange observed by differential titration is therefore the sum of the protons associated with DPG binding at the entrance to the central cavity and the “Bohr protons” which are bound elsewhere (Perutz et al., 1969; Kilmartin and Rossi-Bernardi, 1969). Measurements of the change in oxygen affinity with pH (Benesch et al., 1969a; Tomita and Riggs, 1971; Riggs, 1971) involve a M eren t parameter associated with DPG binding, i.e., the increased strength of binding of DPG to deoxyhemoglobin with increasing H+ concentration. The shape of the curve relating DPG concentration to pb0is therefore p H dependent (Fig. 14,A). As a result ( Alog pE0)/ A ~ H is also made up of two components, i.e., the true “Bohr effect” as well as the DPG shift due to tighter binding at lower pH. This second component increases to a maximum at low DPG concentration and then reaches zero when enough DPG is present to eliminate the pH dependence of DPG binding ( Fig. 14,B).

1.1

-

A

::

a

-

0 0

5

10

15

(DPG) x 1 0 4

20

25

0.6

-

1

I

1

I

I

5

10

15

20

25

[DPG] x 1 0 4

FIG. 14. Effect of 2,3-diphosphoglyceric acid (DPG) on the Bohr coefficient. ( A ) Relation between p~ and DPG concentration at two pH values. ( B ) Change of log p5; with pH between pH 7.3 and 7.6 as a function of DPG concentration. M in 0.05 M bis( 2-hydroxyethyl)iminotris(hyHemoglobin concentration 6 x droxymethyl)methane, 0.1 M C1- temperature 20°C. Data from Benesch et a2. ( 1969a).

230

RUTH E. BENESCH AND REINHOLD BENESCH

The identity of the Bohr coefficients in the absence and in the presence of excess DPG again confirms the conclusion that the protons which participate in the Bohr effect involve sites quite different from the polyphosphate binding one. IV. DPG AND CO, TRANSPORT Bauer was the first to discover (Bauer, 1969,1970) that DPG competes with oxygenation linked CO, binding. Rossi-Bernardi and his collaborators (Pace et al., 1970; Rossi-Bernardi et al., 1972) then found that the differential CO, uptake between oxy- and deoxyhemoglobin is halved when DPG is present. This result, together with the finding that DPG does not affect CO, binding by liganded hemoglobin, agrees remarkably well with the realization (Kilmartin and Rossi-Bernardi, 1971) that CO, forms carbamates both at the and /3 N-terminal NH, groups, but only the latter is competitive with DPG. (Y

V. EFFECXOF DPG ON ABNORMALHEMOGLOBINS The recognition of the DPG binding site in HbA facilitates the interpretation of the effect of organic phosphate on abnormal hemoglobins. Hemoglobin variants in which the amino acid residues in the binding site are not affected and which have a normal oxygenation curve show the same decrease in oxygen affinity in the presence of DPG as hemoglobin A. This is, for example, the case with HbS ( p - 6 Glu+ Val) and HbC (p-6 Glu + Lys) as well as HbA, (Bunn and Briehl, 1970). As discussed earlier, when crucial residues in the binding site are either missing or substituted (HbF, sheep Hb, cat BHb, HbAIC,HbF,) the DPG effect is greatly reduced or absent. In the case of Hb Shepherds Bush (p-74-Gly + Asp) the weaker response to DPG has been ascribed to a disturbance of Lys p-82 by the side chain of the abnormal aspartic acid in the deoxy conformation ( May and Huehns, 1972). Very recently, Bromberg et al. (1973) have described a diminished helcotropic effect of DPG in Hb Little Rock. In this abnormal hemogIobin histidine p-143 which, as will be recalled, is replaced by serine in Hb F, is substituted by a glutamine. In a series of other abnormal hemoglobins, the situation is somewhat more subtle. These are variants with amino acid substitutions in locations which do not involve the DPG binding residues. In such hemoglobins the affinity for organic phosphates is lowered because the abnormality leads to a displacement of the normal equilibrium between the oxy and deoxy conformation (R and T states) in favor of the former.

231

ORGANIC PHOSPHATES AND HEMOGLOBINS

TABLE 111 Binding of Spin-Labeled Triphosphule (SLTP) to Dcoxyhemoglobins*

Hemoglobin HbA azAPzA Hb Chesapeake a2**Arg'Leu&A Hb Kempsey azA8PAsp*Asn ff2"Bz

azSz+CN a

Stoichiometry (moles/ tetramer ) 1.0 1.0 1.0 1.0 1.0

[Hb][SLTP] = [HbSLTP]

2.5 x 7.2 X 1.7 x 6.3 x 3 x

1 0 - 5 ~ 1W6 M 10-4~ 10-5~ 10-4~

Data from Ogata and McConnell (1971, 1972a) and Ogata et al. (1972).

This was conclusively demonstrated by Ogata and McConnell in their study of hemoglobins Chesapeake ( a-92 Arg 3 Leu) and Kempsey (p-99 Asp+ Asn) (Ogata and McConnelI, 1972a; Ogata et QZ., 1972). The affinity constants of the spin-labeled triphosphates for these hemoglobins are considerably lower than for HbA (Table 111). Nevertheless, the spectra of the label bound to these abnormal hemoglobins are essentially identical to those of the HbA complexed label. Since this spectrum is very sensitive to changes in the protein conformation in the vicinity of the label, the binding site per se cannot be responsible for the weakened binding. Therefore, the weaker binding must be due to a reduced concentration of the T state. By application of their Generalized Concerted Transition Model, the authors concluded that the change to the quaternary T state in these hemoglobins is inhibited because their n and p subunits, respectively, are locked in the tertiary r conformation. The high ligand affinity of hemoglobins Chesapeake, Yakima (p-99 Asp + His), and Kempsey was also interpreted by Davis ef al. (1971) in terms of preferential ligand binding by the hemes of the abnormal chains. When the penultimate tyrosine of the p subunits is substituted as in hemoglobins Bethesda ( p 145Tyr 4His) and Rainier ( /3 145Tyr + Cys) the stability of the deoxy conformation is also impaired. The increased oxygen affinity is again accompanied by a diminished helcotropic effect of DPG (Hayashi and Stamatoyannopoulos, 1972). Olson and Gibson (1972b) used dissociation into ap dimers, the kinetics of CO binding and Soret spectra as criteria for the conformational state of hemoglobin Bethesda. All the evidence showed that the substitution of histidine for tyrosine 145p drastically alters the ability of this hemoglobin to assume a deoxy conformation. Only in the presence of organic phosphates, particularly IHP, can this mutant undergo the transition to the quaternary deoxy structure.

232

RUTH E. BENESCH AND REINHOLD BENESCH

Methemoglobins M are mutants in which the iron of either the LY or /3 chain hemes is fixed in the ferric state due to substitutions in the heme pocket, i.e., remote from the polyphosphate binding site. Most of these (Po?Val 3 hemoglobins have a low oxygen affinity. In HbMMileRllhee Glu), the oxygen affinity is further lowered by DPG or IHP (Udem et al., 1970). Ligand binding is cooperative in the absence of the cofactor. In its presence cooperativity is lost, and the ligand is, therefore, bound without a change in conformation. This is borne out by spectroscopic evidence. When the ferrous hemes combine with ligand in the absence of cofactor, a spectral change in the ferrihemes is observed which is not seen in the presence of DPG or IHP. It was therefore concluded that in the presence of phosphates this hemoglobin remains in the T state even when it is fully liganded ( Perutz et al., 1972). VI. ORGANIC PHOSPHATES AND ARTIFICIALHEMOGLOBIN HYBRIDS Valence hybrids in which either the (Y or the /3 hemes are fixed in the ferric state as the cyanide complex (cyanomet forms) have been used as models for intermediates in ligand binding by hemoglobin in several laboratories. However, as pointed out by Ogata and McConnell ( 1971), it should be bowe in mind that “While the hybrid hemoglobins are ‘partially liganded,’ they may not be structurally equivalent to the corresponding partially liganded ferrous tetramers. In addition, hemoglobin molecules of this type-where only a or /3 subunits are liganded-may not actually occur during cooperative ligand binding to ferrous hemoglobin. Therefore, the structures of the hybrids may not be directly relevant to the cooperative phenomenon.” Direct binding measurements by Ogata and McConnell (1971) with their spin-labeled triphosphate led to the results summarized in Table 111. It is clear that the binding of 1 mole per tetramer is much tighter to the hybrid with ferrous /3 chains than to that with ferrous a chains. The invariance of the electron spin resonance (ESR) spectra of the bound label again led to the conclusion that the constants in Table I11 reflect the proportion of R and T state in the deoxygenated hybrids. This is consistent with the results of earlier work on the nuclear magnetic resonance (NMR) spectra of these hybrids as well as kinetic and equilibrium measurements. In the absence of phosphates the NMR spectrum of the ferric portion is unaffected by ligand binding to the ferrous hemes. From the ligandinduced spectral changes in the presence of organic phosphates, it was concluded that the j3 chains are much more effective in the transition to the T state than the chains (Ogawa and Shulman, 1971). Thus the P ferro hybrid undergoes transformation to the T state with DPG and even

ORGANIC PHOSPHATES AND HEMOGLOBINS

233

inorganic phosphate, but the ferro hybrid requires the much more tightly bound IHP for the same transformation (Cassoly et al., 1971). The biphasic kinetics of CO binding also illustrate the existence of two states of different reactivity in these hybrids (Cassoly et al., 1971). The equilibrium between the fast and slowly reacting forms is progressively shifted in favor of the latter by inorganic phosphate, DPG and IHP, i.e., in the order of the affinity of these compounds for the deoxy conformation. Furthermore, less DPG is required to transform the p ferro hybrid into the slowly reacting form, i.e., the T state, than the ferro hybrid. Oxygen equilibrium measurements on the hybrids (Haber and Koshland, 1971; Maeda et al., 1972) also led to similar conclusions, i.e., that the helcotropic effect of DPG is greater when the B , hemes are ferrous. (Y

(Y

VII. SUMMARYAND CONCLUSIONS The realization that organic phosphates participate in the oxygenation of hemoglobin has led to some drastic revisions in our thinking about how hemoglobin functions as an oxygen carrier. First of all, it is now clear that human hemoglobin without organic phosphates binds oxygen far too tightly to release it under physiological conditions. The large intracellular concentration of DPG lowers the oxygen affinity into the useful range, where it is further modulated by organic phosphates, hydrogen ions and CO,. The red cell DPG level is subject to metabolic control, including feedback mechanisms which respond to oxygen requirement. On a molecular level, the basis for the lowering of the oxygen affinity by organic phosphates is their mole for mole interaction with a single site on deoxyhemoglobin which is destroyed on ligand binding. Therefore, the overall free energy of oxygenation is lowered by the binding energy. The binding site in the deoxy tetramer is at the entrance to the central cavity between the terminal ends of the two /3 chains. DPG and similar polyanions bind electrostatically to the cluster of positive amino acid residues which are located there. The similarity of the conclusions on the details of the interaction between DPG and hemoglobin, which were reached from studies in solution and from those on the intact crystal, is indeed impressive. The linkage of phosphate cofactors with ligand binding has also added a new dimension to studies on the mechanism of oxygen transport by hemoglobin. Models for the mechanism of oxygenation must obviously take account of the influence of these cofactors. Thus, for instance, the suggestion that (YP dimers can fulfill the functional properties of hemoglobin (Antonini, 1967; Guidotti, 1967; Antonini and Brunori, 1970) can now be ruled out, since in such fragments the DPG binding site of the

234

RUTH E. BENESCH AND REINHOLD BENESCH

tetramer is gone. From an experimental point of view, the specific stabilization of the deoxy conformation by organic phosphates has already provided a powerful tool for investigations on the conformational transitions which are basic for the understanding of the mechanism of oxygenation.

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