Mixed gelation theory. Kinetics, equilibrium and gel incorporation in sickle hemoglobin mixtures.

This paper outlines a theoretical formalism for describing the gelling behavior of sickle cell hemoglobin in mixtures with other hemoglobin and non-hemoglobin proteins. Experimental applications are reported for hybridized and unhybridized mixtures of HbS (sickle hemoglobin), HbA (adult hemoglobin), HbF (fetal hemoglobin), and HbC Harlem. The theory is a general one based on a modification of the sol—gel phase equilibrium equation to take into account the varying tendencies of different hemoglobin species to promote gelation, and specific hemoglobin interactions are encoded in gelling coefficients which quantify gelling capability. Gelling coefficients for the hemoglobin species dealt with here are evaluated by measuring incorporation into the polymer phase in S-A, S-F, and S-C_H mixtures. Given this information, the theory is found to provide accurate prodictions for the equilibrium gelling behavior of the calibrating pairs themselves when they are hybridized or unhybridized, for gelation kinetics in diverse mixtures of these species taken two, three and four at a time, for the anomalous equilibrium and kinetic gelling behavior of A- C_H mixtures, and it also accounts for a variety of results previously published by others. Apparently, given the gelling coefficients for any mutant hemoglobin, one can compute gelling behavior (equilibrium, kinetics, incorporation, etc.) in any specified mixture with any other known hemoglobin(s). The gelling coefficients for any mutant hemoglobin depend upon, and therefore provide information about, gel interactions at the mutant site. From the gelling coefficients one can also obtain the change in free energy of interaction in the gel due to the altered residue. Experimental approaches are described which allow an analysis for the gelling coefficients of any mutant hemoglobin to be performed in a few hours.     

Maintenance of hemoglobin inducibility in somatic cell hybrids of tetraploid (2S) mouse erythroleukemia cells with mouse or human fibroblasts.

It has previously been reported that Friend mouse erythroleukemia (MEL) cells synthesize hemoglobin when exposed to 2% dimethylsulfoxide, and that hybrids between MEL cells and fibroblasts (or other nonerythroid cells) do not synthesize hemoglobin. We have been successful in obtaining hybrids (3/15) between MEL cells and mouse L-cell fibroblasts that maintain hemoglobin inducibility by preserving nonadherent cells after fusion. The proportion of hemoglobin inducible hybrids can be increased (8/11) by using a stable 2S (pseudotetraploid) MEL parent in addition to preserving nonadherent cells after fusion. All hybrids which were nonadherent were hemoglobin inducible, and all hybrids which were adherent were not. Five nonadherent hybrid clones were analyzed from fusions between a stable 2S MEL parent and a human fibroblast (WI-38, VA-2). All these clones were inducible for hemoglobin. It is concluded that gene dosage is effective in increasing the proportion of hemoglobin inducible hybrids, but hybrid morphology is the phenotype characteristic that correlates most closely with expression of hemoglobin inducibility.     

R-state hemoglobin bound to heterotropic effectors: models of the DPG, IHP and RSR13 binding sites

We performed a docking study followed by a 500-ps molecular dynamics simulation of R-state human adult hemoglobin (HbA) complexed to different heterotropic effectors [2,3-diphosphoglycerate (DPG), inositol hexaphosphate (IHP), and 2-[4-[(3,5-dichlorophenylcarbamoyl)-]methyl]-phenoxy]-2-methylpropionic acid (RSR13)) to propose a molecular basis for recently reported interactions of effectors with oxygenated hemoglobin. The simulations were carried out with counterions and explicit solvation. As reported for T-state HbA, the effector binding sites are also located in the central cavity of the R-state and differ depending on effector anionic character. DPG and IHP bind between the alpha-subunits and the RSR13 site spans the alpha1-, alpha2- and beta2-subunits. The generated models provide the first report of the molecular details of R-state HbA bound to heterotropic effectors.     

The crystal structure of bar-headed goose hemoglobin in deoxy form: the allosteric mechanism of a hemoglobin species with high oxygen affinity

The crystal structure of a high oxygen affinity species of hemoglobin, bar-headed goose hemoglobin in deoxy form, has been determined to a resolution of 2.8 A. The R and R(free) factor of the model are 0.197 and 0.243, respectively. The structure reported here is a special deoxy state of hemoglobin and indicates the differences in allosteric mechanisms between the goose and human hemoglobins. The quaternary structure of the goose deoxy hemoglobin shows obvious differences from that of human deoxy hemoglobin. The rotation angle of one alphabeta dimer relative to its partner in a tetramer molecule from the goose oxy to deoxy hemoglobin is only 4.6 degrees, and the translation is only 0.3 A, which are much smaller than those in human hemoglobin. In the alpha(1)beta(2) switch region of the goose deoxy hemoglobin, the imidazole ring of His beta(2)97 does not span the side-chain of Thr alpha(1)41 relative to the oxy hemoglobin as in human hemoglobin. And the tertiary structure changes of heme pocket and FG corner are also smaller than that in human hemoglobin. A unique mutation among avian and mammalian Hbs of alpha119 from proline to alanine at the alpha(1)beta(1 )interface in bar-headed goose hemoglobin brings a gap between Ala alpha119 and Leu beta55, the minimum distance between the two residues is 4.66 A. At the entrance to the central cavity around the molecular dyad, some residues of two beta chains form a positively charged groove where the inositol pentaphosphate binds to the hemoglobin. The His beta146 is at the inositol pentaphosphate binding site and the salt-bridge between His beta146 and Asp beta94 does not exist in the deoxy hemoglobin, which brings the weak chloride-independent Bohr effect to bar-headed goose hemoglobin.     

Lysine-specific gingipain K and heme/hemoglobin receptor HmuR are involved in heme utilization in Porphyromonas gingivalis

We have previously reported on the identification and characterization of the Porphyromonas gingivalis A7436 strain outer membrane receptor HmuR, which is involved in the acquisition of hemin and hemoglobin. We demonstrated that HmuR interacts with the lysine- (Kgp) and arginine- (HRgpA) specific proteases (gingipains) and that Kgp and HRgpA can bind and degrade hemoglobin. Here, we report on the physiological significance of the HmuR-Kgp complex in heme utilization in P. gingivalis through the construction and characterization of a defined kgp mutant and a hmuR kgp double mutant in P. gingivalis A7436. The P. gingivalis kgp mutant exhibited a decreased ability to bind both hemin and hemoglobin. Growth of this strain with hemoglobin was delayed and its ability to utilize hemin as a sole iron source was diminished as compared to the wild type strain. Inactivation of both the hmuR and kgp genes resulted in further decreased ability of P. gingivalis to bind hemoglobin and hemin, as well as diminished ability to utilize either hemin or hemoglobin as a sole iron source. Collectively, these in vivo results further confirmed that both HmuR and Kgp are involved in the utilization of hemin and hemoglobin in P. gingivalis A7436.     

CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin.

The effects of pH upon infrared spectra [CO stretching frequency (vco) region] and visible spectra of the CO complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A are reported. The vco for leghemoglobin--CO complexes was 1947.5 cm-1 at neutral pH. At acid pH myoglobin-- and hemoglobin--CO complexes developed vco bands at 1966--1968 cm-1, whereas leghemoglobin--CO complexes developed vco bands at approximately 1957 cm-1. All pKapp co values determined by pH-dependent variation of vco fell in the range 4.0--4.6. The pKapp co values determined from visible spectra were consistent with vco-determined values except for that of myoglobin--CO (visible pKapp co = 5.8). The pKapp co values in the 4.0--4.6 range appear to be pK values of the distal histidines, while the visible pKapp co of myoglobin--CO appears to be the pK of a group other than the distal and proximal histidines. The data are consistent with a model in which protonation of the distal histidine permits protein-free heme FeCO geometry in leghemoglobin--CO complexes but not in myoglobin-- or hemoglobin--CO complexes. Thus the heme pockets of leghemoglobins appear to be more flexible than the heme pockets of myoglobin and hemoglobin. The effects of pH upon visible spectra of the O2 complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A also are reported. pKapp o2 values of approximately 5.5 (leghemoglobins) and 4.4 (hemoglobin) are probably the pK values of the distal histidines. Comparisons of pKapp o2 values with pKapp co values indicate a more flexible heme pocket in leghemoglobins than in hemoglobin. The O2 complex of leghemoglobin c2 differed significantly from the O2 complexes of leghemoglobins a and c1 in visible spectra and titration behavior. These differences might be associated with the small structural differences in the region between the E and F helixes of leghemoglobins.     

Description of electrophoretic and chromatographic hemoglobin profile of Rhinoclemmys punctularia

Studies of the hemoglobin pattern in Brazilian reptiles are important for determining ecological and phylogenetic relationships, but they are scarce. Peripheral blood samples were obtained from 7 males and 18 females of Rhinoclemmys punctularia. The hematological profile was based on the total hemoglobin and hematocrit values. The hemoglobin profile was obtained using electrophoretic procedures at different pH, isoelectric focusing, globin chain electrophoresis, and HPLC. The hematocrit (31 +/- 2%) and total hemoglobin (7.5 +/- 0.2 g/dL) values did not indicate gender variations. Alkaline pH electrophoresis of the total blood samples treated with 1% saponin demonstrated the presence of four well-defined hemoglobin fractions, one major component (fraction I), showing cathodic migration and three others faster than fraction I with anodic migration. When the samples were precipitated with chloroform, only two hemoglobin fractions were observed, similar to fractions I and III from the first procedure. Isoelectric focusing and HPLC showed the same pattern. With acid and neutral pH electrophoresis, two fractions with anodic migration were observed. The globin chain identification at alkaline pH showed two fractions, but four fractions were observed at acidic pH, suggesting that different polypeptide chains are involved in the hemoglobin molecule. The chromatographic separation of the total blood sample demonstrated that the major fraction comprised 81.9% and the minor 18.1%. The results obtained demonstrated a similarity between these hemoglobin components and those of some Chelidae reported in the literature for both land and aquatic animals, reflecting the adaptation to environmental conditions.     

Bioelectrocatalytic detection of glycated hemoglobin (HbA1c) based on the competitive binding of target and signaling glycoproteins to a boronate-modified surface

We developed an electrochemical glycated hemoglobin (HbA(1c)) biosensor for diagnosing diabetes in whole human blood based on the competitive binding reaction of glycated proteins. Until now, no studies have reported a simple and accurate electrochemical biosensor for the quantification of HbA(1c) in whole blood. This is because it is very difficult to correctly distinguish HbA(1c) from large amounts of hemoglobin and other components in whole blood. To detect glycated hemoglobin, we used electrodes modified with boronic acid, which forms a covalent bond between its diol group and the cis-diol group of the carbohydrate moiety of glycated proteins. For accurate HbA(1c) biosensing, we first removed blood components (except for hemoglobin) such as glycated proteins and blood glucose as they interfere with the boronate-based HbA(1c) competition analysis by reacting with the boronate-modified surface via a cis-diol interaction. After hemoglobin separation, target HbA(1c) and GOx at a predetermined concentration were reacted through a competition onto the boronate-modified electrode, allowing HbA(1c) to be detected linearly within a range of 4.5-15% of the separated hemoglobin sample (HbA(1c)/total hemoglobin). This range covers the required clinical reference range of diabetes mellitus. Hence, the proposed method can be used for measuring %HbA(1c) in whole human blood, and can also be applied to measuring the concentration of various glycated proteins that contain peripheral sugar groups.     

The deoxygenation kinetic properties of human fetal hemoglobin: effect of 2,3-diphosphoglycerate

The deoxygenation kinetics of isolated adult and fetal hemoglobin are measured. The results demonstrate that significant functional differences exist between the two tetrameric hemoglobins. It is pointed out that these functional differences closely parallel the differences in similar properties of beta and gamma chains. It is also shown that 2,3-diphosphoglycerate (2,3-DPG) has no significant effect on the deoxygenation rate of fetal hemoglobin. This result appears to be consistent with the reported weaker binding of 2,3-DPG to the oxygen linked groups of fetal hemoglobin.     

Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence.

The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.     

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.     

Polymeric nanoparticles for hemoglobin-based oxygen carriers

This article reports on the current status of the research on blood substitutes with particular attention on hemoglobin-based oxygen carriers (HBOCs). Insights on the physiological role of hemoglobin are reported in the view of the development of both acellular and cellular hemoglobin-based oxygen carriers. Attention is then focused on biocompatible polymeric materials that find application as matrices for cellular based HBOCs and on the strategies employed to avoid methemoglobin formation. Results are reported regarding the use of bioerodible polymeric matrices based on hemiesters of alternating copolymer (maleic anhydride-co-butyl vinyl ether) for the preparation of hemoglobin loaded nanoparticles.     

The Electrophoretic Pattern of Hemoglobin in Newborn Babies,and Abnormalities of Hemoglobin F Synthesis in Adults

On routine electrophoretic analyses on filter paper and starch gel in an alkaline or neutral medium, no abnormal hemoglobin fractions were found in the blood of 600 newborn infants or their mothers. Trace amounts of hemoglobin Barts were noted in many of the blood samples from newborns when the starch gels (phosphate buffer pH 7.0) were stained with a benzidine/H₂O₂ reagent. In one infant, precocious cessation of synthesis of hemoglobin F was postulated to account for the small amounts of this hemoglobin found in a cord-blood specimen. Analysis of 15,000 blood samples from adults revealed two instances in which the hemoglobin F level was 20 and 35%, respectively. The former was attributed to a hereditary persistence of hemoglobin F, while the latter was associated with acute leukemia.In an addendum, the finding of an infant with an abnormal hemoglobin variant, resembling in many of its properties hemoglobin F Texas, is reported.     

Studies on the characteristics of alginate gels in relation to their use in separation and immobilized applications

Studies on diffusion of NAD and hemoglobin from calcium and barium gels are reported where alginate grade, concentration, and gel dimensions were varied. These show that NAD diffusion characteristics are unaffected by alginate and ion concentrations; however, hemoglobin diffusion is affected by alginate concentration. Both hemoglobin and NAD diffusion patterns were shown to be affected by alginate gel dimensions. Studies are reported that show that polymannuronic alginate gels posses good porosity characteristics while polyguluronic alginates from gels with lower porosity, specifically with respect to high-molecular-weight compounds. These findings are discussed with the view to the use of alginate gels for immobilization, solids separation, and diffusion chromatography techniques.     

Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii

The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues. The N-terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.     

Effect of temperature on oxygen affinity and anion binding of bovine hemoglobin

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.     

Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins

A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.     

Oxidation-reduction potentials of human fetal hemoglobin and gamma chains. Effects of blocking sulfhydryl groups.

The oxidation-reduction equilibrium of the gamma chains of human fetal hemoglobin (Hb F) has been studied and compared with that of the alpha and beta chains of human adult hemoglobin (Hb A). The effects of the sulfhydryl (--SH) reagents, iodoacetate, iodoacetamide, and p-mercuribenzoate (PMB), on the three kinds of chains and on Hb F have been compared. The midpoint potentials (E-m) of all three sorts of chains are lower than those of tetrameric hemoglobin A or F. The E-m values of alpha chains are the lowest, E-m = 0.049 volt at 6 degrees, and are unaffected by pH change or by PMB treatment, at least from pH 6 to 8. The E-m values of beta-SH chains are higher; E-m = 0.102 volt at pH 7, decreasing to 0.050 volt at pH 8, both at 6 degrees. These results agree with those of Banerjee and Cassoly ((1969) J. Mol. Biol. 42, 337-349). They reported no effect of PMB on beta chains, but we find that 2 eq of PMB/chain raise E--M to 0.139 volt at pH 7 at 6 degrees, chiefly as the result of reaction at beta-93, not at beta-112. Carboxymethylation at beta-93 has an insignificant effect compared with that of PMB. The oxidation-reduction potential of gamma chains is similar to that of beta chains. E-m = 0.098 volt at pH 7 at 6 degrees, decreasing to 0.064 at pH 8 and 0.010 at pH 9. The effects of --SH reagents, reacting at position gamma-93 (the only --SH group present in gamma chains), are essentially the same as those seen with beta chains. The oxidation-reduction potential of Hb F is almost identical with that of Hb A, except for being 0.008 volt lower at pH 6 at 6 degrees. This agrees with the results reported by Flohe and Uehleke ((1966) Life Sci. 5, 1041-1045). PMB or iodoacetamide treatment lowers E-m by 0.02 to 0.03 volt, depending on the pH, from 6 to 9, in much the same way as previously reported for Hb A(Brunori, M., Taylor, J.F., Antonini, E., Wyman, J., and Rossi-Fanelli, A. (1967) J. Biol. Chem. 242, 2295-2300). The "residual oxidation Bohr effect" noted in Hb F can be attributed to the oxidation Bohr effect of the gamma chains. The apparent pK of the heme-linked water molecule was found at 25 degrees to be, for Hb F, 8.1; for gamma-SH chains, 7.85; for gamma-PMB chains, 8.35; and for gamma chains treated with iodoacetate, 7.80. Sedimentation coefficients, s-20, w, at a protein concentration of 5 mg/ml, were found to be, for fetal hemoglobin 4.09, for iodoacetamide-treated fetal hemoglobin 4.04, for PMB-treated fetal hemoglobin 3.41, for fetal gamma-SH chains 4.25, and for fetal gamma-PMB chains 3.08.     

Further resolution of adult chick hemoglobins by isoelectric focusing in polyacrylamide gel.

Evidence is presented that adult chick hemoglobins exist in four types separable by isoelectric focusing on polyacrylamide gels instead of the two hemoglobin types previously resolved by other methods. These are hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin D1 (HbD1), and hemoglobin D2(HbD2). Their pI values are 7.53 +/- 0.02, 7.37 +/- 0.02, 6.92 +/- 0.04 and 6.72 +/- 0.05, respectively, constituting about 63, 14, 18 and 5% of the total hemoglobin from adult chick erythrocytes, respectively. HbA1 and HbA2 ar identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weight and amino acid composition of HbD1 and HbD2 are also identical although there are differences in their tryptic peptides. Experiments were done to show that the existence of four hemoglobin types is not due to genetic heterogeneity of the experimental animal, nor to artifacts of oxidation of carboxyhemoglobin to methemoglobin tetramers. Care was exercised to eliminate deamination and modification of side chain amino groups by using freshly prepared hemolysates and to minimize the "plateau phenomenon" peculiar to isoelectric focusing by controlling the duration of electrophoresis. The use of cyanmet form of (thus liganded) hemoglobin in this study reduced the chance of heterotetramer formation. Furthermore, consideration was given to possible anomalies caused by ampholytes. In the face of negative evidence for artifacts, it is concluded that adult chicken has more than the two hemoglobin types previously reported.     

The effect of 2,3-diphosphoglycerate on the tetramer-dimer equilibrium of carbon monoxide hemoglobin in dilute solution. Correlation between sedimentation and kinetic behavior

The effect of 2,3-diphospho-D-glycerate on the sedimentation coefficient of carbon monoxide hemoglobin was correlated with the fraction of rapidly reacting hemoglobin observed subsequent to flash photolysis at 23 degrees C at pH 7.30 in buffers of 0.1 M ionic strength. Concentrations of the organic phosphate up to about 5 mM resulted in an increase in S20,w, consistent with an increase in the fraction of tetrameric hemoglobin. A decrease in rapidly reacting hemoglobin parallelled the increase in the sedimentation coefficient. Between 5 and 20 mM 2,3-diphosphoglycerate, S20,w decreased, suggesting that dissociation to dimers was enhanced. An increase in rapidly reacting hemoglobin was also observed in this concentration range. Similar sedimentation results were obtained with oxyhemoglobin at pH 7.00 and carbon monoxide hemoglobin at pH 7.06. Assuming single binding sites on each species, the dissociation constants for 2,3-diphosphoglycerate binding to tetrameric and dimeric HbCO are 0.2-0.3 mM and 2-5 mM at pH 7.30. This biphasic effect of this physiologically important organic phosphate on the state of aggregation of R state hemoglobin has not been previously reported, but it is similar to that previously noted with inositol hexaphosphate, which enhanced tetramer formation at low concentrations, while at higher concentrations it promoted hemoglobin dissociation to dimers (White, S. L. (1976) J. Biol. Chem. 251, 4763-4769; Gray, R. D. (1980) J. Biol. Chem. 255, 1812-1818).