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In the human hemoglobin variant Hb Hinsdale, lysine is substituted for asparagine at position beta 139 (H17), which lies in the water-filled cavity that runs through the center of the molecule. This substitution adds two extra cationic residues to the excess of four cationic residues normally lining this cavity. Moo-Penn and colleagues who discovered this hemoglobin, found its oxygen affinity in 0.5 M bis-Tris buffer to be lower than that of Hb A. Their finding conflicted with our prediction that additional cationic groups lining the central cavity would destabilize the T-structure by increased electrostatic repulsion and thereby increase the oxygen affinity. We have, therefore, remeasured the ligand-binding properties of Hb Hinsdale. In chloride-free Hepes buffer, Hb Hinsdale has greatly increased oxygen affinity and lower cooperativity than Hb A. A comparison of the properties of Hb A, Hb Hinsdale, Hb Deer Lodge (beta 2 His-->Arg) and Hb Abruzzo (beta 143 His-->Arg) in 0.05 M Hepes versus 0.05 M bis-Tris buffers shows that very low chloride concentrations can significantly alter cooperativity as well as oxygen affinity. The apparent conflict between the findings of Moo-Penn and colleagues and our prediction arises from the enhanced chloride effects exhibited by Hb Hinsdale. On going from 0.05 M Hepes to 0.05 M bis-Tris at pH 7.0, log P50 values for Hb A and Hb Hinsdale are increased by 0.28 and 1.12, respectively. The Bohr effect, the kinetics of oxygen dissociation, the second-order rate constant of CO binding and the rate of CO recombination after flash photolysis were also determined for Hb Hinsdale. The enhanced chloride sensitivity of Hb Hinsdale is consistent with the allosteric mechanism of chloride interactions proposed by Perutz et al. in the accompanying paper.

Hemoglobin Catonsville is a mutation of human hemoglobin (an alpha 2 beta 2 tetramer) in which a glutamate residue is inserted into the first turn of a highly conserved 3(10) helix (the C helix) of each alpha subunit. In theory, amino acid insertions (or deletions) in protein helices can be accommodated via two distinct mechanisms. One, termed the register shift mechanism, preserves the geometry of the helix while requiring all of the residues on one flank of the insertion site to rotate by 100 degrees in the case of an alpha helix or by 120 degrees in the case of a 3(10) helix. The other, termed the bulge (or indentation) mechanism, distorts the local geometry of the helix but does not alter the helix register. High-resolution X-ray diffraction analysis of deoxyhemoglobin Catonsville shows that the inserted residue is accommodated as a bulge, demonstrating that this is a viable mechanism. (In contrast, no such evidence is yet available for the register shift mechanism.) More specifically, the insertion converts one turn of the C helix from 3(10) geometry to alpha helix-like geometry, raising the possibility that a common mechanism for accommodating insertions and deletions within helices may involve localized interconversions between 3(10), alpha, and pi helical structures.

Hb Rancho Mirage was detected in a 17-year-old male in association with a mild anemia. Hemoglobin electrophoresis revealed the variant had a mobility between Hbs A and J on cellulose acetate (pH 8.6) and a mobility like Hb F on citrate agar (pH 6.4). A substitution of His----Asp was found at position 143 in the beta chain, a residue that contributes to the anionic 2,3-DPG binding site in Hb. This variant exhibited normal oxygen affinity at physiologic pH and reduced affinity at alkaline pH. This suggested a subtle shift in the allosteric equilibrium due most likely to the introduction of a negative charge that stabilized the 2,3-DPG pocket. Both homotrophic (heme-heme) and heterotropic (2,3-DPG and protons) effects were reduced; this might be a consequence of an alteration in the carboxyl terminal region of the beta-subunits. Although a His----Asp substitution would be considered to cause reasonable disruption of the 2,3-DPG and C-terminal conformation of the beta- subunits, the properties of Hb Rancho Mirage suggest that, in fact, there appear to be no major perturbation of the critical C-terminal residues.

A family was studied who carried a slow mobility haemoglobin on cellulose acetate electrophoresis at pH 8.6. The structural analysis of the anomalous globin chain showed substitution of residual aspartic acid in position 94 of the alpha chain by tyrosine (Hb Setif). This mutation induces low oxygen affinity in the haemoglobin molecule plus instability of the tetramer in the oxy conformation. Such haemoglobin has been found in North-African populations, and the case presented here is the first one reported in Spain.

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.

Hemoglobin Tampa was detected in a 6-year-old male caucasian who is homozygous for this variant hemoglobin. The variant hemoglobin has an electrophoretic mobility between Hb F and Hb S on cellulose acetate (pH 8.5) and a mobility between Hb S and Hb C on citrate agar (pH 6.0). In acid buffer globin chain analysis revealed an abnormal beta chain with a mobility between the beta A and beta S chains, and in alkaline buffer the mobility of the chain was at the beta S position. Structural characterization of the variant beta chain indicates that aspartic acid is replaced with tyrosine at position 79, the site of a previously reported mutation, Asp replaced by Gly (Hb Hsi-Tsou). The clinical histories of the available family members including the homozygous propositus appear to be unremarkable.

The evaluation of a family with chronic mild anemia led to the identification of a new unstable hemoglobin (Hemoglobin Cheverly). Modest anemia and reticulocytosis, normal to slightly increased mean corpuscular volume (MCV), and normal mean corpuscular hemoglobin concentration (MCHC) were present in the affected family members. Electrophoresis of blood samples on cellulose acetate and on citrate agar revealed normal patterns. Globin chain analysis and isoelectric focusing data were also normal. After incubation for 3 h at 41 degrees C, Heinz bodies were detected in 95-100% of erythrocytes from affected individuals. Positive heat and isopropanol tests confirmed the initial observation of the Heinz body preparation and indicated that an unstable hemoglobin was present. Structural analysis showed an amino acid substitution of Phe-Ser at position 45 (CD4) in the beta chain. Hemoglobin Cheverly has a reduced affinity for oxygen and a reduced Bohr effect, properties that can be rationalized on the basis of the x-ray crystallographic structure of normal hemoglobin. Despite structural and functional similarities between Hb Cheverly and Hb Hammersmith, beta 42 (CD1) Phe-Ser, the clinical manifestations of Hb Cheverly are mild in contrast to the severe disease observed with Hb Hammersmith. Reasons for the apparently silent clinical expression of Hb Cheverly are not known. We discuss the implications of unstable hemoglobins in the evaluation of chronic anemia in pediatric patients.

Hb Evanston (alpha 14 Trp leads to Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A2 suggested the presence of a variant Hb A2 with a substitution in the alpha-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than 10% occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the alpha-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of Hb Evanston with alpha-thalassemia-2 produced anemia in this black family.

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated alpha-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 (P 1/2 = 0.60 and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect (-delta log P 1/2/pH (7 to 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 X 10(6) M-1 S-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 X 10(6) M-1 S-1), whereas Hb Wayne Asp showed heterogeneous binding (l' = 6.1 X 10(6) and 2.6 X 10(6) M-1 S-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.

Hemoglobin Fannin-Lubbock was found in a 9-year-old Mexican-American female. The abnormal hemoglobin was detected as a fast-moving variant by electrophoresis on cellulose acetate at pH 8.4. Structural analysis indicated a substitution in the beta-chain of aspartic acid for glycine at position 119, a position involved in the alpha1beta1 contact of the hemoglobin tetramer. This contact between unlike chains is larger and undergoes a smaller shift during the process of oxygenation and deoxygenation that the alpha1beta2 contact (Perutz, M.F., Muirhead, H., Cox, J.M. and Goaman, L.C.G. (1968) Nature 219, 131-139). Mutations in this contact tend to cause slight or no changes in functional behavior. Apart from a mild anemia, the propositus did not exhibit any obvious clinical symptoms.

Our studies on the separation of hemoglobin variants using Monobead ion exchangers show that these ion exchangers will resolve variants that heretofore could not be resolved. Procedures with these ion exchangers, therefore, extend our ability to detect hemoglobinopathies. Since it is also important to study the physicochemical properties of variants, their isolation is of critical importance to the researcher. The methods described in this report enable variants to be isolated in quantitative amounts so that such studies can be performed. Most isolations can be accomplished within 1 h, under mild conditions with aqueous buffers. Elution gradients can be changed conveniently to optimize resolution of most variants. These methods should serve as a valuable adjunct in studying the structure-function relationship of hemoglobin and other proteins.

Three anticoagulant proteins were isolated from the venom of Naja nigricollis (spitting cobra). The peaks of anticoagulant activity co-chromatographed with phospholipase A2 activities. The three proteins were homogeneous by the criteria of electrophoresis in two acidic polyacrylamide gel systems. Electrophoresis in sodium dodecyl sulfate also yielded single protein bands with molecular weights of about 15,000. The amino acid compositions of the three anticoagulant proteins are reported. All three proteins have only one amino acid replacement in the first 25 to 30 amino acids of their NH2-terminal sequences, compared to the sequence of the basic phospholipase from N. nigricollis venom. Removal of Ca2+ from the crude venom caused loss of both anticoagulant and phospholipase activities, and restoration of Ca2+ caused partial recovery of both activities. Both activities were lost in parallel when the venom was heated between pH 6.0 and 8.5. The association of the anticoagulant effect with phospholipase activity contradicts the previous conclusion that phospholipase is not responsible for the anticoagulant action of cobra venom. The previous results might be explained by independence of the anticoagulant and phospholipase effects within the same protein molecule, or by different activity levels of monomer and dimer forms of the enzyme.

Hemoglobin Queens: alpha 34 (B15) Leu-Arg was found in association with Hb E in a Vietnamese boy. The observed microcytosis and hypochromia were due to Hb E, which also occurred in other members of this family. The functional properties of Hb Queens in the absence and presence of allosteric effectors were normal. The abnormal hemoglobins appear to be unrelated to the hyperbilirubinemia that was observed at birth.

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.

Hemoglobin Shelby, detected in two unrelated black families, has an electrophoretic mobility like Hb F on cellulose acetate (pH 8.4) and a mobility between Hbs S and C on citrate agar (pH 6.2). Globin chain analysis in acid and alkaline buffers revealed an abnormal chain migrating between beta A and beta S. Tests for unstable hemoglobins were positive. Hematologic data on both families indicated carriers have mild anemia. The variant showed a slightly lower affinity for oxygen with normal cooperativity and Bohr effect, and its reactions with 2,3-diphosphoglycerate and inositol hexaphosphate were similar to those of Hb A. Sequence analysis indicated the substitution of lysine for glutamine at position 131 in the beta-chain. In a previous report (1) we described a variant, Hb Deaconess, in which this residue was deleted. On reexamination of the data, we find that Hb Deaconess is identical to Hb Shelby.