Characteristic of aromatic amino acid substitution at alpha 96 of hemoglobin

Replacement of valine by tryptophan or tyrosine at position alpha96 of the alpha chain (alpha96Val), located in the alpha(1)beta(2) subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the alpha96 position. The characteristic of aromatic amino acid substitution at the alpha96 of hemoglobin has been further investigated by producing double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp). r Hb (alpha42Tyr --> Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between alpha42Tyr and beta99Asp in thealpha(1)beta(2) subunit interface of deoxy Hb A. The second mutation, alpha96Val -->Trp, may compensate the functional defects of r Hb (alpha42Tyr --> Phe), if the stability due to the introduction of trypophan at the alpha 96 position is strong enough to overcome the defect of r Hb (alpha42Tyr --> Phe). Double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb (alpha42Tyr --> Phe). (1)H NMR spectroscopic data of r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between alpha 42Tyr and beta 99Asp is essential for the novel oxygen binding properties of deoxy Hb (alpha96Val --> Trp) .     

Hemoglobin S Travis: a sickling hemoglobin with two amino acid substitutions [beta6(A3)glutamic acid leads to valine and beta142 (h20) alanine leads to valine).

Hb S Travis is a previously undescribed sickling hemoglobin with two amino acid substitutions in the beta chain: beta6 Glu leads to Val and beta142 Ala leads to Val. The beta6 Glu leads to Val mutation imparts to Hb S Travis the characteristic properties of sickling hemoglobin, namely its association with erythrocyte sickling, the insolubility of the hemoglobin in the reduced form, and a minimum gelling concentration value identical to Hb S. Unlike Hb S, Hb S Travis exhibits an increased oxygen affinity and a decreased affinity for 2,3-bisphosphoglycerate and inositol hexakisphosphate. In addition, the variant hemoglobin's tendency to autoxidize and its mechanical precipitability suggest that there are conformational differences between Hb S and Hb S Travis.     

Hemoglobin Wood beta97(FG4) His replaced by Leu. A new high-oxygen-affinity hemoglobin associated with familial erythrocytosis.

The characterization of hemoglobin Wood (beta97(FG4) His replaced by Leu), a high oxygen affinity hemoglobin with reduced Hill constant is described. The amino acid substitution occurs at the alpha1beta2 interface, in the same position as in hemoglobin Malm? (beta97(FG4) His replaced by Gln) and in an homologous position when compared with hemoglobins Chesapeake (alpha92(FG4) Arg replaced by Leu) and J. Capetown (alpha92(fg4) arg replaced by Gln).     

N-terminal contributions of the γ-subunit of fetal hemoglobin to its tetramer strength: Remote effects at subunit contacts

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the gamma-subunit of fetal hemoglobin, which differs from the beta-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the gamma-subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the beta-subunit by the corresponding residues in the gamma-subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (gamma) of HbF with its high pK(a) of 8.1 compared with a 7.1 value for Val 1 (beta) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the gamma-subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (beta) by Glu 5 (gamma) promotes tetramer strengthening. The Glu --> Asp replacement at position 7 enhances this effect because of the lower pK(a) of Asp but the Val --> Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.     

Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the beta 27 (B9) position

We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.     

Proton nuclear magnetic resonance studies of hemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKee Rocks (beta145HC2 Tyr replaced by term): an assignment for an important tertiary structural probe in hemoglobin

High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.     

Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.     

Mechanical precipitation of hemoglobin k?ln.

Hb K?ln (beta 98 Val leads to Met) was found to precipitate rapidly during mechanical shaking. The rate of precipitation of Hb K?ln is 5-6 times faster than that of Hb S. The kinetics of precipitation of the patient's hemolysate, which is a mixture of Hb K?ln and Hb A, showed a biphasic curve indicating that Hb K?ln precipitates independently from Hb A. The instability of Hb K?ln may be attributed to the conformational change in the vicinity of heme. The mechanical shaking may be used as a new method for detection and quantitation of hemoglobin K?ln and other unstable hemoglobins.     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Allosteric interactions in non-alpha chains isolated from normal human hemoglobin, fetal hemoglobin, and hemoglobin Abruzzo (beta143 (H21) His replaced by Arg).

Oxygen-linked effects of inositol hexaphosphate occur in heme-containing non-alpha chains isolated from normal human hemoglobin, fetal hemoglobin, and the abnormal human hemoglobin Abruzzo, beta143(H21) His leads to Arg. The occurrence of these effects implies that the chains undergo ligand-linked conformational changes. Inositol hexaphosphate lowers the oxygen affinity of isolated beta and gamma chains by differential binding to their deoxy conformations. Neither 2,3-diphosphoglycerate nor inorganic phosphate produces such an effect. In the case of Abruzzo beta chains, the binding of inorganic phosphate and 2,3-diphosphoglycerate is also oxygen-linked. Stripped beta chains isolated from hemoglobin Abruzzo have much higher oxygen affinity than beta chains isolated from HbA. Their higher oxygen affinity and enhanced allosteric interactions with phosphates account, in large part, for the abnormal functional behavior of the hemoglobin Abruzzo tetramer. In this hemoglobin variant the substitution of arginine for histidine at beta143 involves a residue known to interact with anionic allosteric effectors of hemoglobin. It is of interest that the effect of inositol hexaphosphate observed with isolated gamma chains is comparable to the effect observed with isolated beta chains, even though the gamma143 position is occupied by an uncharged serine residue.     

Introduction of a new regulatory mechanism into human hemoglobin

Previous studies on bovine hemoglobin (HbBv) have suggested amino acid substitutions, which might introduce into human hemoglobin (HbA) functional characteristics of HbBv, namely a low intrinsic oxygen affinity regulated by Cl(-). Accordingly, we have constructed and characterized a multiple mutant, PB5, [beta(V1M + H2 Delta + T4I + P5A + A76K)] replacing four amino acid residues of HbA with those present at structurally analogous positions in HbBv, plus an additional substitution, beta T4I, which does not occur in either HbBv or HbA. This 'pseudobovine' hemoglobin has oxygen binding properties very similar to those of HbBv: the P(50) of HbA, PB5 and HbBv in the absence of Cl(-) are 1.6, 4.6 and 4.8 torr, respectively, and in 100 mM Cl(-) are 3.7, 10.5 and 12 torr, respectively. Moreover, PB5 has 3-fold slower autoxidation rate compared to HbA and HbBv. These are desirable characteristics for a human hemoglobin to be considered for use as a clinical artificial oxygen carrier. Although the functional properties of PB5 and HbBv are similar, van't Hoff plots indicate that the two hemoglobins interact differently with water, suggesting that factors regulating the R to T equilibrium are not the same in the two proteins. A further indication that PB5 is not a functional mimic of HbBv derives from PB5(control), a human hemoglobin with the same substitutions as PB5, except the beta T4I replacement. PB5(control) has a high oxygen affinity (P(50)=2.3 torr) in the absence of Cl(-), but retains the Cl(-) effect of PB5. The Cl(-) regulation of oxygen affinity in PB5 involves lysine residues at beta 8 and beta 76. PB4, which has the same substitutions as PB5 except beta A76K, and PB6, which has all the substitutions of PB5 plus beta K8Q, both have a low intrinsic oxygen affinity, like HbBv and PB5, but exhibit a decreased sensitivity to Cl(-). Since HbBv has lysine residues at both beta 8 and beta 76, these results imply that Cl(-) regulation in HbBv likewise involves these two residues. The mechanism responsible for the low intrinsic oxygen affinity of HbBv remains unclear. It is suggested that residues peculiar to HbBv at the alpha(1)beta(1) interface may play a role.     

Crystal structure of Lysbeta(1)82-Lysbeta(2)82 crosslinked hemoglobin: a possible allosteric intermediate

The crystal structure of human hemoglobin crosslinked between the Lysbeta82 residues has been determined at 2.30 A resolution. The crosslinking reaction was performed under oxy conditions using bis(3, 5-dibromosalicyl) fumarate; the modified hemoglobin has increased oxygen affinity and lacks cooperativity. Since the crystallization occurred under deoxy conditions, the resulting structure displays conformational characteristics of both the (oxy) R and the (deoxy) T-states. beta82XLHbA does not fully reach its T-state conformation due to the presence of the crosslink. The R-state-like characteristics of deoxy beta82XLHbA include the position of the distal Hisbeta63 (E7) residue, indicating a possible reason for the high oxygen affinity of this derivative. Other areas of the molecule, particularly those thought to be important in the allosteric transition, such as Tyrbeta145 (HC2) and the switch region involving Proalpha(1)44 (CD2), Thralpha(1)41 (C6) and Hisbeta(2)97 (FG4), are in intermediate positions between the R and T-states. Thus, the structure may represent a stabilized intermediate in the allosteric transition of hemoglobin.     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.     

Receptor tyrosine kinase signaling required for integrin alpha v beta 5- directed cell motility but not adhesion on vitronectin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.     

Role of Bohr group salt bridges in cooperativity in hemoglobin.

Possible problems in measuring the first Adair constant, K1, from accurate oxygen equilibrium curves have been investigated. Of these only the presence of small amounts of CO-hemoglobin or hemoglobin dimers had a significant effect. The former can be eliminated by treatment with oxygen, the latter by measuring the concentration-dependence of K1 or working at high protein concentrations. K1 values have been measured for normal hemoglobin at pH 7 and 9, hemoglobin specifically reacted with cyanate at Val 1alpha (alphac2beta2) and des(His 146beta) hemoglobin at pH 7. K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7. This shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT. Hence the Bohr group salt bridges break on ligation of the T state and are partially responsible for the free energy of cooperativity.     

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malm? (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.     

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.     

Three-point cross-linking: potential red cell substitutes from the reaction of trimesoyl tris(methyl phosphate) with hemoglobin.

The symmetrical trifunctional cross-linking reagent trimesoyl tris(methyl phosphate) (3), reacts selectively with amino groups (beta 1Val and beta 82Lys) in the diphosphoglycerate binding site of human hemoglobin A, producing cross-linked tetrameric species in good yield. A major species is triply linked, alpha alpha beta 1(82) greater than B beta 82, where B symbolizes benzene-1,3,5-tricarbonyl. Both this triply linked species and the doubly linked species, alpha alpha beta 1B beta 82, produced from deoxyhemoglobin have a considerably lower oxygen affinity than does native hemoglobin while maintaining a high degree of cooperativity (n50 = 2.4), making them potentially useful as red cell substitutes, in principle delivering twice as much oxygen as whole blood between pO2 = 100 and = 40 Torr. The yield of products indicates that triply and doubly linked species form in parallel so that there are independent routes to each. It is proposed that differences in routes are due to stereoisomerism about the amide bonds which form from reaction of the reagent with the protein.     

Specific antibodies to hemoglobin A1 (anti-Glu) and hemoglobin S (anti-Val) in the guinea pig: immunologic and structural correlations.

Like goats and sheep, guinea pigs can produce, in response to human sickle cell hemoglobin (beta6 Glu leads to Val), an antibody population (anti-Val) that will bind sickle cell hemoglobin but not normal hemoglobin HbA. Unlike goats and sheep, guinea pigs can produce in response to human hemoglobin A1 an antibody fraction, anti-Glu, that will not react with human sickle cell hemoglobin. These anti-Glu antibodies have been isolated by affinity chromatography and their specificity confirmed by fluorescence-quenching titrations. The sequence of the first 10 amino acids of the beta-chain of guinea pig hemoglobin has been determined. This sequence differs from those of both hemoglobin HbA and sickle cell hemoglobin by two residues, those at positions 5 and 6. This explains the similarity of the immunogenicity of this site on the two human hemoglobins when administered to guinea pigs. Both goats and sheep are identical to hemoglobin A1 at the beta-6 position.     

Hemoglobin Brockton [beta 138 (H16) Ala----Pro]: an unstable variant near the C-terminus of the beta-subunits with normal oxygen-binding properties

Hemoglobin Brockton [beta 138 (H16) Ala----Pro] is an unstable variant associated with a mild anemia. It has the same electrophoretic mobility as and cannot be resolved from Hb A. Oxygen affinity measurements of blood and hemolysate do not indicate biphasic oxygen saturation, showing that the functional properties of the variant are very similar to those of Hb A. This implies that the introduction of proline into the H-helix at position 138 does not disrupt the critical inter- and intrasubunit hydrogen bonds and salt bridges at the beta carboxyl-terminal dipeptide, since these polar interactions are essential for the normal oxygen-binding properties of hemoglobin. X-ray crystallographic data are consistent with these findings and show that the consequences of the beta 138 Ala----Pro substitution are almost entirely confined to the immediate vicinity of the mutation site. Instability probably results from the inability of a buried hydrogen bond to form between Pro 138 beta and Val 134 beta.