Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.     

Molecular cloning and structural characterization of the hagfish proteinase inhibitor of the alpha-2-macroglobulin family

The most primitive living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human ₂-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg⁸³³) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg⁸³³ was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.     

Front Cover Image,Volume 117, Number 1, January 2020

Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, −80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and β subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O₂ equilibrium curve, O₂ dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.     

The stability of the heme-globin linkage in some normal, mutant, and chemically modified hemoglobins.

The rates and equilibria of heme exchange between methemoglobin and serum albumin were measured using a simple new spectrophotometric method. It is based on the large difference between the spectrum of methemoglobin and that of methemealbumin at pH 8-9. The rate of heme exchange was found to be independent of the albumin concentration and inversely proportional to the hemoglobin (Hb) concentration. Taken together with the finding that the rate was 10 times greater for Hb Rothschild, which is completely dissociated into alpha beta dimers and 10 times smaller for two cross-linked hemoglobins, the subunits of which cannot dissociate, this showed that the rate of dissociation of heme from alpha beta dimers is very much greater than from tetramers. Conditions were found for the attainment of an equilibrium distribution of hemes between beta globin and albumin. The equilibrium distribution ratio, R = methemealbumin/albumin/methemoglobin/apohemoglobin, for hemoglobin A was 3.4 with human and 0.005 with bovine serum albumin. Both the rates of exchange and the R values of HbS and HbF were the same as that for HbA. The equilibrium distribution ratio for Hb Rothschild was 7 times greater than that for HbA whereas that of one but not the other of the cross-linked hemoglobins was 10 times smaller. The structural bases for these differences are analyzed.     

The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. II. Influence of the intersubunit contact splitting

The rate of the H-D exchange of the peptide NH atoms of the isolated alpha and beta subunits of human Hb were studied at the pH range 5.5-9.0 and 20 degrees C by the IR spectroscopy. The factor retardation of the exchange rate of subunits -P in the range -10(2)-10(7). In comparison with tetramer Hb the probability of local fluctuations (1/P) is increased to a slightly greater extent for the monomeric alpha subunits then for the tetramer beta subunits. Unlike Hb oxygenation of subunits does not influence on the probability of the local fluctuations and subunits have no the pH-dependent change of the value 1/P observable for the ligand Hb. The possible mechanisms of the overall intensification of the local fluctuations upon the splitting of the Hb tetrameric contacts between subunits are discussed with the inviting of the structural crystallographic data.     

Dynamic nature of the quaternary structure of the vesicular stomatitis virus envelope glycoprotein

The envelope glycoprotein (G protein) of vesicular stomatitis virus probably exists in the viral envelope as a trimer of identical subunits. Depending on the conditions of solubilization, G protein may dissociate into monomers. G protein solubilized with the detergent octyl glucoside was shown to exist as oligomeric forms by sedimentation velocity analysis and chemical cross-linking. G protein was modified with either fluorescein isothiocyanate or rhodamine isothiocyanate. Resonance energy transfer between fluorescein and rhodamine labels was observed upon mixing the two labeled G proteins in octyl glucoside. This result provided further evidence that G protein in octyl glucoside is oligomeric and indicated that the subunits are capable of exchange to form mixed oligomers. Resonance energy transfer was independent of G protein concentration in the range examined (10-80 nM) and was not observed when labeled G proteins were mixed with fluorescein or rhodamine that was not conjugated to protein. Resonance energy transfer decreased upon incorporation of G protein into Triton X-100, consistent with sedimentation velocity data that G protein in Triton X-100 is primarily monomeric. Kinetic analysis showed that the subunit exchange reaction had a half-time of about 3 min at 27 degrees C that was independent of G protein concentration. These data indicate that the exchange occurs through dissociation of G protein trimers into monomers and dimers followed by reassociation into timers. Thus, in octyl glucoside, G protein must exist as an equilibrium between monomers and oligomers. This implies that monomers are capable of self-assembly into trimers.     

Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethyl-porphyrinatoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using (19)F NMR and the O(2) binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in alpha- and beta- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O(2) affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O(2) affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O(2) affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.     

Isolation and characterization of a protein from hagfish serum that is homologous to the third component of the mammalian complement system.

The 192-kDa protein HX, a major component of serum that specifically binds to zymosan particles, was prepared from the plasma of the hagfish (Eptatretus burgeri) by ion-exchange chromatography and gel filtration. HX, present at a concentration of 0.8 mg/ml in the original plasma, was composed of two distinct subunits of 115 kDa and 77 kDa, respectively, which were linked by disulfide bonds. The protein had the same electrophoretic mobility as beta-globulin. Digestion by trypsin resulted in a specific cleavage of the 115-kDa subunit and a change in its immunoelectrophoretic mobility in the anodal direction, leaving the 77-kDa subunit intact. Treatment with SDS and urea resulted in the splitting of the 115-kDa subunits into 68-kDa and 45-kDa components, but this splitting was inhibited by pretreatment with methylamine, suggesting the presence of a thiol ester bond in the 115-kDa subunit. The amino acid composition of HX revealed a striking resemblance to that of human C3. We conclude, therefore, that the 192-kDa protein isolated in this study is analogous to C3, which plays a key role in the mammalian C system.     

Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions

We compared oxygenation and anaerobic oxidation reactions of a purified complex of human hemoglobin (Hb) and haptoglobin (Hb-Hp) to those of uncomplexed Hb. Under equilibrium conditions, Hb-Hp exhibited active-site heterogeneity and noncooperative, high-affinity O(2) binding (n(1/2)=0.88, P(1/2)=0.33mm Hg in inorganic phosphate buffer at pH 7 and 25°C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O(2) dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb-Hp had significantly reduced absorption at the λ(max) of 430nm relative to uncomplexed Hb, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E(1/2)) of Hb-Hp was found to be +54mV, showing that it is much more easily oxidized than uncomplexed Hb (E(1/2)=+125mV). The Nernst plots for Hb-Hp oxidation showed no cooperativity and slopes less than unity indicated active-site heterogeneity. The redox potential of Hb-Hp was unchanged by pH over the range of 6.4-8.3. Exposure of Hb-Hp to excess hydrogen peroxide (H(2)O(2)) produced ferryl heme, which was found to be more kinetically inert in the Hb-Hp complex than in uncomplexed Hb. The negative shift in the redox potential of Hb-Hp and its stabilized ferryl state may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb-Hp complex.     

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.     

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)     

Angiotensin I-converting enzyme-like activity in tissues from the Atlantic hagfish (Myxine glutinosa) and detection of immunoreactive plasma angiotensins

Using a highly sensitive fluorimetric assay, significant levels of angiotensin I -converting enzyme-like activity (ACELA) were detected in a range of tissues (branchial heart, gill, kidney with associated vasculature and archinephric duct, liver, whole brain and gut) from the Atlantic hagfish (Myxine glutinosa). The highest ACELA occurred in heart and gill (1.8 and 1.5 nmol His–Leu min⁻¹ mg protein⁻¹, respectively). The mammalian angiotensin I-converting enzyme (ACE) inhibitor, captopril, at 10⁻⁵ M was a potent inhibitor of the ACELA found in all hagfish tissues. Radioimmunoassay showed that immunoreactive angiotensins (251.8±11.8 pM) were detectable in hagfish plasma. The validity of the assay for measurement of hagfish angiotensins was indicated by the parallelism of the angiotensin II standard curve against serially diluted hagfish plasma. Measurement of immunoreactive plasma angiotensins and detection of significant levels of ACELA in a wide range of tissues gives indirect evidence for the presence of a renin–angiotensin system in hagfishes, the earliest evolved group of craniates.     

1H NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A

The reaction of heme and apoprotein has been studied in detail in 1H NMR spectroscopy in order to elucidate the conditions for reconstitution of hemoglobin (Hb) to yield the native protein. The initially formed holoprotein exists as a mixture of isomers with individual subunits possessing the two heme orientations differing by a 180 degrees rotation about the alpha, gamma-meso axis [La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., & Pandey, R. K. (1985) Biochemistry 24, 3826-3831]. We characterize in detail herein the rates and mechanism of heme reorientation and show that the rates differ dramatically for met-aquo and met-azido derivatives and are highly pH dependent in both subunits in a fashion that allows selective equilibration in either subunit. Nonequilibrium mixtures of such isomers can be kinetically trapped in the met-azido form and stored in this metastable form for many months. With kinetically controlled heme orientationally disordered Hb, unambiguous assignment of 1H NMR resonances to individual subunits has been made for the met-azido derivative, which demonstrates approximately 2% and 10% equilibrium heme disorder in the alpha- and beta-subunits, respectively. Comparison of the 1H NMR spectra of various heme rotationally disordered Hb derivatives indicates that this disorder is observable in all forms studied, but is most easily recognized as heme disorder and most conveniently monitored in the met-azido complex. Structural consequences of heme disorder appear to manifest themselves much more strongly in peripheral than axial interactions at the heme. Preliminary studies reveal that both the rate of autoxidation of oxy-Hb and the azide affinity of met-aquo-Hb depend on the orientation of the heme.     

Molecular and complex-chemical studies on methemoglobin from Chironomus thummi thummi and various isolated fractions]

1. Six different hemoglobin (Hb) fractions were isolated and characterized from the larvae of Chironomus thummi thummi using column chromatographic procedures. 2. Chromatographic and sedimentation-analytic studies (sedimentation coefficients of 2.0 +/- 0.2 (S)) have shown three Hb fractions to exist basically in a monomeric form. The molecular weight of component M-2 was determined by sedimentation equilibrium technique to be 15,470 +/- 400. The dimeric Hb was found to have sedimentation coefficients of 3.0 +/- 0.1 (S) in the weakly acidic pH region. In alkaline milieu, the reversible dissociation proceeds into the monomeric molecules (S20, W = 1.9 +/- 0.1 (S)). Molecular weights vary between pH 5.7 and 9.8 not only with hydrogen ion concentration, but also with protein concentration in correspondence with a dissociation-association equilibrium consisting of monomers and dimers. 3. For the Hb fraction M-2, a friction ratio of f/fo = 1.03 was calculated, suggesting an almost spherical shape of this protein. In contrast, the dimeric component appears to have a much more asymmetric structure (f/fo = 1.19). 4. The indivdual MetHb fractions bind the ligands: fluoride, imidazole and azide with different affinities.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: 2. Effect of intersubunit contact cleavage

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in isolated α and β subunits of human hemoglobin (Hb) at pH 5.5–9.0 and 20°C. The H-D exchange occurs by the EX2 mechanism. The retardation factor of subunit exchange rate (P) is in a range of approximately 10²–10⁷. Compared to tetrameric Hb, the probability of local fluctuations (1/P) increases to a slightly greater extent in monomeric α subunits than in tetrameric β subunits. Unlike in the whole Hb molecule, oxygenation of its subunits has no effect on the probability of local fluctuations, and the subunits show no pH-dependent changes in 1/P values (observed for liganded Hb). Probable mechanisms accounting for overall intensification of local fluctuations upon the cleavage of contacts between subunits of the tetrameric Hb molecule are discussed with regard to structural crystallographic data.     

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.     

Molecular interactions between ribosomal proteins--a study of S4-S9 interaction.

The ribosomal proteins S4 and S9 were isolated from the 30S ribosomal subunit of Escherichia coli to greater than 95% purity and characterized in the reconstitution buffer. Neither of the proteins indicated any tendency to self associate at 3 degrees C in the concentration range studied. At higher temperatures (greater than 20 degrees C), protein S9 forms a significant amount of a soluble aggregate as seen from the sedimentation velocity and sedimentation equilibrium experiments. From an analysis of the solution mixture of S4 and S9 at 1:1.08 molar concentration ratio by sedimentation velocity experiment, an s20,w value of 1.77 +/- 0.02S was obtained. A fast moving component which accounts for approximately 20% of the mass was also observed. Increasing the concentration of S9 does not alter the observed s20w value significantly for that component which could be followed. A detailed analysis of the data obtained at 3 degrees C from sedimentation equilibrium experiments on mixtures of the proteins indicated that a species of molecular weight greater than either of the two proteins was present. The proteins were found to interact with a mean equilibrium constant of association of 3.66 +/- 2.39 x 10(4) M-1 and a Gibbs free energy of interaction, delta Go = -5.8 kcal/mole at 3 degrees C in TMKD buffer. This information helps in understanding the energetics of the 30S ribosomal subunits of E. coli.     

Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces

Different types of human hemoglobins (Hbs) consisting of various combinations of the embryonic, fetal, and adult Hb subunits are present at certain times during development representing a major paradigm of developmental biology that is still not understood and one which we address here. We show that the subunit interfaces of these Hbs have increasing bonding strengths as demonstrated by their distinct distribution of tetramers, dimers, and monomers during gel filtration at very low‐Hb concentration. This maturation is mediated by competition between subunits for more favorable partners with stronger subunit interactions. Thus, the protein products of gene expression can themselves have a role in the developmental process due to their intrinsic properties.     

Antagonistic effect of urea on oxygenation-linked binding of ATP in an elasmobranch hemoglobin

The O2 affinity of "stripped" (cofactor-free) hemoglobin (Hb) of the elasmobranch, Squalus acanthias is decreased by ATP, the main erythrocytic phosphate cofactor but increased by urea at physiological concentration. When both compounds are present, as in life, urea decreases the ATP sensitivity, indicating that previous Hb oxygenation studies in the absence of urea overestimate the modulator role of phosphate cofactors in sharks. Whereas ATP decreases the O2 association equilibrium constant of the deoxygenated pigment, urea raises those of both the deoxy and the oxygenated states. Possible mechanisms for the urea-protein interactions i.e. binding at carboxy-termini or carbamylation of amino-termini of the protein chains, are discussed.