Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.     

Isohemoglobin differentiation in the bimodal-breathing amazon catfish Hoplosternum littorale

The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum littorale that frequents hypoxic habitats uses "mammalian" 2,3-diphosphoglycerate (DPG) in addition to "piscine" ATP and GTP as erythrocytic O(2) affinity modulators. Its electrophoretically distinct anodic and cathodic hemoglobins (Hb(An) and Hb(Ca)) were isolated for functional and molecular characterization. In contrast to Hb(An), phosphate-free Hb(Ca) exhibits a pronounced reverse Bohr effect (increased O(2) affinity with decreasing pH) that is obliterated by ATP, and opposite pH dependences of K(T) (O(2) association constant of low affinity, tense state) and the overall heat of oxygenation. Dose-response curves indicate small chloride effects and pronounced and differentiated phosphate effects, DPG < ATP < GTP < IHP. Hb(Ca)-O(2) equilibria analyzed in terms of the Monod-Wyman-Changeux model show that small T state bond energy differences underlie the differentiated phosphate effects. Synthetic peptides, corresponding to N-terminal fragment of the cytoplasmic domain of trout band 3 protein, undergo oxygenation-linked binding to Hb(Ca), suggesting a metabolic regulatory role for this hemoglobin. The amino acid sequences for the alpha and beta chains of Hb(Ca) obtained by Edman degradation and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensitivities.     

Structural, functional, and subunit assembly properties of hemoglobin Attleboro [alpha 138 (H21) Ser----Pro], a variant possessing a site maturation at a critical C-terminal residue

Hemoglobin Attleboro, a new alpha-chain variant with a substitution of proline for serine at position 138 (H21), was found to be a noncooperative high-affinity hemoglobin (P50 = 0.26 mmHg at pH 7 and 20 degrees C) which lacked an alkaline Bohr effect. Addition of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP) led to a decrease in oxygen affinity but to no alteration in either Bohr effect or cooperativity. Ligand binding kinetics studies revealed an overall rate of oxygen dissociation at pH 7.0 and 20 degrees C that was 2.7-fold slower than that for Hb A. At pH 8.5, the kinetic profile was identical with that at pH 7, confirming the absence of a Bohr effect for this variant hemoglobin. Measurement of the rate of oxygen dissociation with carbon monoxide replacement indicated a lack of cooperativity. Sedimentation velocity experiments yielded s20,w values of 2.8 and 4.3 for 65 microM solutions of oxyhemoglobins Attleboro and A, respectively (indicating an enhancement in the oxy dimer population of this variant). Studies of the carbon monoxide combination of this variant revealed an association rate 20-fold faster than that for Hb A; only in the presence of a 1000-fold molar excess of IHP was there a significant reduction in the overall rate. Rapid-scan and traditional stopped-flow experiments conducted in the Soret Soret region demonstrated an alteration in the structure and rate of assembly of the deoxy tetramer of Hb Attleboro relative to that of Hb A. The abnormal properties of this hemoglobin variant can be attributed to major perturbations in the C-terminal region.     

Structural and functional studies of Hemoglobin Wayne: An elongated α-chain variant

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated α-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.60}$ and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect ($\text{?Δ}\text{log}\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{/}\text{pH}\text{(7}\text{to}\text{8) = 0.34}$ and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l′ = 5.6 × 10⁶m^?1s^?1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l′ = 2.6 × 10⁶m^?1s^?1), whereas Hb Wayne Asp showed heterogeneous binding (l′ = 6.1 × 10⁶ and 2.6 × 10⁶m^?1s^?1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.     

Structural and functional studies of hemoglobin Barcelona (α2β2 94 Asp (FG1) → his): Consequences of altering an important intrachain salt bridge involved in the alkaline Bohr effect

Hemoglobin Barcelona was discovered by routine electrophoresis in a Spanish family showing a mild polycythemia. Red blood cells of the propositus which contained 37% of the abnormal hemoglobin had an increased oxygen affinity and a lowered alkaline Bohr effect. After purification, functional studies of Hb² Barcelona (pI = 7.11) demonstrated a twofold increase in oxygen affinity and a moderate reduction in heme-heme interaction compared to normal HbA. Its reaction towards anionic cofactors (Cl^?, DPG or IHP) was similar to that of HbA. Reactivity of the sulphydryl groups (cysteine-β93) was increased in Hb Barcelona both in the deoxy and fully liganded forms, and in the absence as well as in the presence of IHP. By three different methods (the pH-dependence of log P₅₀, the direct proton titration technique and the measurement of the ΔpIdeox-ox) by isoelectric focusing) all in the absence of phosphate ions, Hb Barcelona was found to have a 20 to 30% reduction of the alkaline Bohr effect. This was most pronounced in the alkaline pH range. The reduction was less than expected for the loss of the important intrachain salt-bridge Asp-β94 → His-β146 considered to be responsible for 40 to 60% of the whole T → R Bohr effect (Perutz et al., 1980). This suggested that in Hb Barcelona, His-β146 could be in weak electrostatic interaction with the neighboring Glu-β90 in the deoxy form. It is concluded that the presence of the oxygen-linked Asp-β94 → His-β146 salt-bridge in HbA is a prerequisite for the full expression of the alkaline Bohr effect and heme-heme interaction.     

Oxygen transport properties of blood in two different bovine breeds

1. The whole oxygen dissociation curve of oxyhemoglobin has been determined in double-muscled cattle of the Belgian White Blue breed and in Friesian cattle of different body weight. 2. In calves, P50 values are low and DPG level is high (4-20 mumol/g Hb). 3. P50 values of 25 +/- 1.4 mm Hg (mean +/- SD) and a level of DPG less than 1.5 mumol/g Hb have been found in animals weighing more than 80 kg. 4. Effects of temperature and pH on the oxygen dissociation curve have been measured at all levels of saturation. The temperature coefficient (dlog P50/dT) and the Bohr effect expressed as dlog P50/dpH were 0.017 and -0.40, respectively. 5. Hematocrit, hemoglobin concentrations and oxygen capacity of hemoglobin have been measured. 6. No difference between both breeds has been observed. 7. These data can be used to correct measured values of oxygen tension for temperature and pH and to measure oxygen content of blood in cattle.     

Self-association of haemoglobin Olympia (alpha 2 beta 2 20 (B2) Val----Met). A human haemoglobin bearing a substitution at the surface of the molecule

Oxygenation measurements at equilibrium were carried out for solutions of pure haemoglobin (Hb) Olympia (alpha 2 beta 2 20 (B2) Val----Met) at 200 microM (haem) and revealed a high oxygen affinity (P50 = 4.2 torr at pH 7.20, 25 degrees C) compared to HbA (P50 = 5.6 torr), with the Hill coefficient (eta H) reduced from the normal value of 2.9 to 2.5 for Hb Olympia at neutral pH. 2,3-Diphosphoglycerate and chloride effects were normal, but measurements of the alkaline Bohr effect indicated an excess Bohr effect of about 20% for Hb Olympia. Precise determinations of the oxygen binding curves gave the unexpected finding of a dependence of co-operativity on pH with eta H rising from 2.4 at pH 6.8 to 3.0 at pH 8. Moreover, the Hill coefficient was dependent upon the concentration at alkaline pH and fell to 1.8 in low concentration solutions (approximately 30 microM-haem) of the haemoglobin variant; at this concentration the Bohr effect was normal. This effect of concentration on co-operativity could be accounted for fully by the allosteric model, with introduction of Hb Olympia self-association. In this case the allosteric constant L' for the supramolecular species has the value of the allosteric constant L for the tetramer species, raised to a power equal to the number of molecules in the aggregates and modulated by the ratio of the dissociation constants of the aggregates, DNR/DNT. Model curves with N tetramers per aggregate (where N approximately 2 at pH 7.5 and N approximately 4 at pH 8.0) fully represented the concentration dependence for Hb Olympia of the eta H values and the detailed shape of the experimental curves for eta H as a function of log[y/(1-y)], the first derivative of the Hill plot. These curves are much steeper when supramolecular species are present. Direct measurements of the protein aggregation by centrifugation confirmed the presence of aggregates in the solutions of Hb Olympia. Hb Olympia is therefore one of the few examples of mutant human haemoglobins that self-associate with functional consequences in terms of oxygen binding properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hemoglobin providence. Functional consequences of two alterations of the 2,3-diphosphoglycerate binding site at position beta 82.

Position beta 82 in human hemoglobin (Hb) is normally occupied by lysine, a positively charged residue that is involved in the binding of anionic cofactors. This residue is substituted by a neutral residue in Hb Providence Asn and by a negatively charged residue in Hb Providence Asp. Hb Providence Asp shows more differences from Hb A than does Hb Providence Asn in studies of the kinetics and equilibria of ligand binding. For both forms, homotropic (cooperative) interactions are normal with n values of 2.5 to 2.7, while heterotropic (pH and anion) interactions are reduced greatly. The reduction in anion sensitivity is attributed to the absence of a positive residue at position beta 82. Reduction in pH sensitivity may be due to a ligand-linked change in the pK of a neighboring residue, beta 143 histidine, which normally is not a Bohr group. This change in pK would act in opposition to the normal Bohr effect. Reduction in the net positive charge of the central cavity has a further consequence. Relative to Hb A, both Hb Providence Asn and Hb Providence Asp show decreased oxygen affinities at neutral pH in the absence of cofactors. This suggests that in Hb A the binding of anionic cofactors directly influences the oxygen affinity by neutralizing the charged groups of the diphosphoglycerate binding site and thus stabilizing the low affinity (T) conformation. From pH 6 to 9 in the presence of 1 M NaCl, where all the charged groups may be masked, the oxygen-binding properties of Hb A and the Hb Providence mutants are identical. Moreover, subunit dissociation of the liganded Hb Providence mutants appears to be increased, as is known to occur for Hb A in the presence of high salt. The results obtained with Hb Providence Asn and Hb Providence Asp illustrate how single amino acid substitutions can modify hemoglobins' pH and anion interactions without altering cooperative interactions between subunits. The alteration in cofactor effects observed with these mutants also illustrates differences between the allosteric effects induced by organic and inorganic anions.     

Characterization of the hemoglobins of the adult brushtailed possum, Trichosurus vulpecula (Kerr) reveals non-genetic heterogeneity

The hemoglobins contained within the red blood cells of the adult brushtail possum exhibited cooperative (n=2.6) oxygen binding curves with an associated p50 of 38 mm Hg at pH 7.4 and a large Bohr effect (-0.60). Stripped hemolysate showed a Bohr effect of -0.27, and was sensitive to added DPG (K=56 micromol L(-1)), ATP (K=130 micromol L(-1)), and chloride ions. Four isoforms of hemoglobin were identified using isoelectric focusing. Mass spectrometry indicated that all four isoforms most likely represent the same gene products which have differentially undergone post-translational deamidation and glutathionylation. The oxygen binding characteristics of three isolated isohemoglobins have been determined.     

Hemoglobin Deer Lodge (beta 2 His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Deer Lodge is an abnormal human hemoglobin with arginine substituted for histidine at the beta 2 position. X-ray crystallography of normal human hemoglobin has shown that the beta 2 residue is normally part of the binding site for 2,3-diphosphoglycerate. The substitution of arginine for histidine at beta 2 affects both the kinetics and equilibria of ligand binding. When stripped of anions, Hb Deer Lodge has an increased oxygen affinity and a decreased degree of cooperativity relative to Hb A. The alkaline Bohr effect is slightly increased and there are marked increases in oxygen affinity below pH 6 and above pH 8. In the presence of 2,3-diphosphoglycerate the cooperativity in increases to nromal and the pH dependence of oxygen binding is reduced. This contrasts with the enhanced Bohr effect seen for Hb A in the presence of organic phosphates. Due to enhanced anion binding at high pH, Hb Deer Lodge has a slightly lower oxygen affinity than Hb A at pH 9 in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate. Kinetic studies at neutral pH in the absence of organic phosphates revealed biphasicity in the rate of oxygen dissociation from Hb Deer Lodge, while approximately linear time courses were observed for Hb A. The fast phase of the oxygen dissociation kinetics shows great pH sensitivity, and organic phosphates increase the rate and percentage of the fast phase without greatly affecting the slow phase. The two phases are not resolvable at high pH. CO combination kinetics are much like those of Hb A except that "fast" and "slow" phases were apparent at wavelengths near the deoxy-CO isobestic point. We suggest that functional differences between the alpha and beta chains are enhanced in Hb Deer Lodge. After flash photolysis of the CO derivative, the percentage of quickly reacting material was slightly greater for Hb Deer Lodge than for Hb A. This may imply a somewhat greater tendency to dissociate into high affinity subunits. The substitution of arginine for histidine at beta 2 thus results in a macromolecule whose ligand-binding properties are significantly altered, the primary differences being expressed at high pH where Hb Deer Lodge binds anions more strongly than Hb A. The properties of Hb Deer Lodge are compared to those of other hemoglobin variants with substitutions at residues involved in binding of 2,3-diphosphoglycerate.     

Hemoglobin-oxygen affinity in anemia

In blood of 21 anemic patients and 8 normal subjects (N) three oxygen dissociation curves each were measured at different pH values to calculate Bohr coefficients after acidification with CO2 (BCCO2) or fixed acid (BCFA), and other important parameters of oxygen affinity. The patients had either low hemoglobin or red cell production (L: n = 11, 7.3 g/dl Hb) or high erythrocyte production combined with high loss (H: n = 10, 7.8 g/dl Hb). The standard half saturation pressure P50 (pH 7.4, 37 degrees C) was equally elevated in both anemic groups (L: 30.5, H: 30.8, N: 26.7 mmHg), as well as the diphosphoglycerate concentration (DPG) (L: 18.7, H: 18.6, N: 12.7 mumol/g Hb). The red cell pH of the anemics was lower than for the N (approximately 0.045 units) causing part of the difference in P50. Hill's "n" tended to high values in the anemics except at low O2-saturation in the H. For BCCO2 no significant difference among the groups was observed. BCFA, however, increased in the H at low SO2 compared to the N and L. The cause for most of the changes in hemoglobin oxygen affinity in anemics was the high [DPG]. The combination of high P50 and high "n" value as in the L seems to be most advantageous for tissue oxygenation.     

Saturation dependency of the Bohr effect: interactions among H-+, CO2, and DPG.

The Bohr effect was measured in normal whole blood and in blood with low DPG concentration as a function of oxygen saturation. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HC1 at constant PCO2 (fixed acid Bohr effect). At nornal DPG concentration CO2 Bohr effect was -0.52 at 50% blood oxygen saturation, increasing in magnitude at lower saturation and decreasing in magnitude at higher saturation. In DPG depleted blood with base excess (BE) similar to 0 meq/1, there was similar dependence of CO2 Bohr effect on oxygen saturation. At BE similar to -10 meq/1, influence of saturation was comparable, but the magnitude of the Bohr effect was markedly increased at all saturations. Fixed acid Bohr effect at normal DPG concentration was -0.45 at saturations of 50-90% but decreased at lower saturations. In DPG-depleted blood fixed acid Bohr effect averaged about -0.33 with minimal variation with saturation. Influence of DPG on oxygen affinity was greater at intermediate saturations and less at saturations below 20% and above 80%. Effect of CO2, independent of pH, was many fold greater at lower oxygen saturations than at higher saturations. These results support the suggestion that the alpha chain of hemoglobin is the site of the initial oxygenation reaction. Physiologically they indicate that the relative contribution of CO2 and fixed acid, as well as the level of oxygen saturation and DPG concentration, may be important in determining PO2 of capillary blood and resulting oxygen delivery.     

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).     

An expanded two-state allosteric model for interactions of human hemoglobin A with nonsaturating concentrations of 2,3-diphosphoglycerate

Oxygen binding curves (OEC) for red cell suspensions have a biphasic shape and reduced n50 values when the concentration of 2,3-diphosphoglycerate (DPG) is lowered by aging or experimental procedures. The mechanism for the abnormal shape of the OEC has been related to variations in the activity of free DPG. DPG binds to tetrameric Hb at a single site, and in red cells its normal concentration is equivalent to that of tetrameric Hb. This equivalence renders the oxygen affinity of Hb and the shape of the OEC very sensitive to small changes in the activity of DPG. The OEC for stripped Hb solutions in the presence of nonsaturating concentrations of DPG also exhibit a biphasic shape but with much larger changes in the n values than observed for red cells. Upon addition of chloride, a known competitor of DPG binding to Hb, the shape of the OEC becomes similar to that of red cell suspensions with the same DPG/Hb ratio. Studies on Hb solutions in the presence of varying concentrations of DPG, but without chloride, have revealed that the cofactor shifts the entire OEC to the right, including both its upper and lower asymptotes. This finding indicates that DPG lowers the intrinsic oxygen affinity for both the T and R states. Theoretical considerations leading to a successful modeling of OEC obtained under varying conditions of DPG and chloride require an expanded two-state allosteric model in which allowance is made for DPG-dependent variations in the dissociation constants of oxygen for both the T and R conformations.     

Hemoglobins Austin and Waco: two hemoglobins with substitutions in the alpha 1 beta 2 contact region.

Hemoglobins (Hbs) Austin and Waco were detected by their electrophoretic migration on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). By these methods, both variants migrated between Hbs A and F. Globin chain analysis at pH 8.6 indicated that the mutant β chain of Hb Austin was faster moving than the β^A chain; however, the mutant chain of Hb Waco was indistinguishable from the β^A chain by this technique. The two variants were isolated by ion-exchange column chromatography. Sequence studies demonstrated a substitution of serine (Hb Austin) and lysine (Hb Waco) for arginine at position 40 in the β chain. These mutations involve an amino acid residue in the α₁β₂ contact region, which, before this report, had been considered invariant in all hemoglobin sequences. Hb Austin was found to exist as dimers when oxygenated and as tetramers when deoxygenated. The equilibrium constant (K_d) for the tetramer to dimer transition was approximately 300 × 10^?6m, as calculated from sedimentation velocity studies. This variant also had high oxygen affinity, a much reduced heme-heme interaction, and a normal Bohr effect. The functional properties of Hb Waco were similar to those of Hb A.     

The effect of the immobilization of hemoglobin on its oxygen binding]

Hemoglobin (Hb) covalently fixed to CM-Sephadex was found to bind oxygen in weakly acidic medium with higher affinity than free Hb. The opposite relation is seen in the alkaline pH region. The alkaline Bohr effect was determined to be -0.2 only. Cooperativity is pH dependent. The sigmoid coefficient at pH 6 is 0.7; at pH 8.7 n was determined to be 1.3. As the reason of these altered binding properties a blockade of the primary amino groups, disturbance of the salt bridges, and restrained cooperative mobility of the Hb-subunits are discussed. The Hill coefficient is additionally lowered by the heterogeneity of the immobilized Hb.     

A follow-up on functional parameters of blood stored in ACD at +4 degrees C

The main functional parameters of blood stored at +4 degrees C in ACD, according to the common transfusional practice, have been carefully followed in the course of 40 days. The expected depletion of DPG takes place within 10 days, but apparently, no increase of the Hb affinity towards oxygen is observed in this period (or later), because pH lowering acts in the opposite direction during the same time. However, the intrinsic increased affinity of Hb is promptly revealed if the "actual" pHs are corrected at the standard value of 7.4, and/or are extrapolated at this pH from Bohr effect.     

Involvement of Glu G3(101)beta in the function of hemoglobin. Comparative O2 equilibrium studies of human mutant hemoglobins

The glutamyl residue at G3(101)beta of normal hemoglobin (Hb A) is one of the alpha 1 beta 2 subunit contacts which are vital to O2 binding properties of the molecule. The O2 equilibrium properties of the four mutants with different substitutions at this site are studied in order to elucidate the role of this residue. Under stripped conditions with minimum chloride the order of O2 affinity is: Hb A (Glu) much less than Hb Rush (Gln) less than or equal to Hb British Columbia (Lys) less than or equal to Hb Potomac (Asp) less than or equal to Hb Alberta (Gly). The first Adair constants, K1, for the mutant hemoglobins are greater than that for Hb A whereas the fourth, K4, are similar, indicating that the allosteric constants (L) of these mutants are greatly reduced. Therefore, the G3(101)beta residue contributes intrinsically to the strengthening of the structural constraints that are imposed upon the deoxy (T) forms but not the oxy (R) form. On addition of 0.1 M Cl- and further addition of 2,3-diphosphoglycerate or inositol hexaphosphate, their O2 affinities and cooperativities are altered, reflecting different responses to anionic ligands. Hb Rush exhibits a stronger chloride effect than Hb A and the other variants and, as a result, an increased Bohr effect and a smaller heat of oxygenation at pH 6.5. These changes are consistent with an increased positive net charge in the central cavity of Hb Rush and subsequent extra anion binding in the deoxy form. The tetramer to dimer dissociation constants are estimated to be greater than normal for Hb British Columbia and less than normal for Hb Alberta. This comparative study of the G3(101)beta mutants indicates that the size and the charge of this residue may influence the switching of two neighboring interchain hydrogen bonds that occurs during oxygenation of normal hemoglobin.     

Measurement of the binding of antifibrinolytic amino acids to various plasminogens

A method for studying the binding of various antifibrinolytic amino acids to plasminogen has been devised. This method is based upon the ability of inhibitors of the streptokinase-induced conversion of plasminogen to plasmin to produce an alteration in the s_20,w⁰ of native plasminogen accompanying their binding to plasminogen. Typical examples of antifibrinolytic amino acids, e.g., 6-amino hexanoic acid, trans-4-aminomethyl cyclohexane-1-carboxylic acid, and l-lysine cause alterations in the s_20,w⁰ of streptokinase-insensitive plasminogens as well as streptokinase-sensitive plasminogens from 5.1–5.6 S to 4.1–4.7 S depending upon the particular plasminogen used. Titration of the s_20,w⁰ of human plasminogen (streptokinase-sensitive) using absorption optics in the analytical ultracentrifuge with the above three compounds led to dissociation constants of 4.5 ± 0.8 × 10⁻⁴m, 8.0 ± 0.8 × 10⁻⁵m, and 6.8 ± 0.8 × 10⁻²m, respectively. When duck plasminogen (streptokinase-insensitive) was used, dissociation constants of 5.6 ± 0.7 × 10⁻⁴m, 9.0 ± 0.8 × 10⁻⁵m, and 8.8 ± 0.7 × 10⁻²m, were obtained.     

Reduced hemoglobin affinity for oxygen in venous blood following hemodilution, independent of changes in pH or PCO2.

A rapidly induced and readily reversible shift in the affinity of hemoglobin for oxygen has been demonstrated. The shift, similar to the Bohr effect, is independent of PCO2 or pH changes. It occurred within 30 min of hemodilution and was seen in portal venous blood but not arterial blood. A hypothesis is suggested involving a phasic alteration in levels of 2,3-diphosphoglycerate (DPG) or ATP binding to hemoglobin. It is proposed that, following hemodilution, the degree of these phosphates to hemoglobin increases on passage through the intestinal vascular bed. The increased DPG binding to hemoglobin results in displacement of additional oxygen. As the blood becomes reoxygenated, the levels of DPG-hemoglobin binding decline and DPG is displaced from the hemoglobin by oxygen.