Characterization of a new electrophoretically silent hemoglobin variant. Hb saale OR alpha 2beta 2 84(EF8)Thr --> Ala

A new abnormal hemoglobin was detected in a young German anemic patient by cation-exchange high performance liquid chromatography (HPLC). Using a combination of electrospray mass spectrometry, HPLC, direct sequencing, and family screening with polymerase chain reaction/restriction digestion approach, we have characterized this hemoglobin variant as resulting from a Thr --> Ala replacement at beta84(EF8). It could be separated neither by electrophoresis nor by isoelectric focusing. Hb Saale is slightly unstable, exhibiting a moderate tendency to auto-oxidize. Functional properties and the heterotropic interactions are similar to those of Hb A.     

Hemoglobin J Cairo: beta 65 (E9) Lys leads to Gln, A new hemoglobin variant discovered in an Egyptian family.

The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found. The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine beta 65 is replaced by glutamine. The new Hb mutant is designated hemoglobin J Cairo beta 65 (E9) Lys leads to Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.     

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.     

A new abnormal human hemoglobin: Hb Prato (alpha 2 31 (B12) Arg leads to Ser beta 2).

An abnormal human hemoglobin was found in a hemolysate from a 5-year-old healthy child living in Prato (Tuscany, Italy). Strutctural studies demonstrated a previously unreported amino acid substitution, alpha 31 (B12) Arg leads to Ser (this is an alpha 1 beta 1 contact). The new variant has been named Hb Prato. It was unstable in isopropanol and heat-denaturation tests, but has normal functional properties, with respect to whole blood studies. Family studies indicated that the variant had been inherited from the mother, a 39-year-old woman of Sicilian extraction. Hb Prato occurs at 20 and 28% in hemolysates from the boy and woman, respectively.     

Clinicohematologic effects of hemoglobin Altdorf (alpha 2 beta 2 135 Ala replaced by Pro)

Hb Altdorf alpha 2 beta 2 135 Ala leads to Pro is an unstable variant occurring near Lecce in Italy. The abnormal hemoglobin does not separate from Hb A in the electrophoresis. In vitro a marked Heinz body formation is produced with phenylhydrazin. In heterozygous individuals an almost compensated hemolysis and a slight splenomegaly are found. Hemolysis can be aggravated by exogenous factors. A rather severe hemolysis was induced by a viral infection in a 3 years old girl.     

Three cases of hemoglobin O Arab.

Three new cases of Hb 0 Arab in two families are reported from districts of Bulgaria, where a carrier state of this abnormal hemoglobin has not been established so far. One of the propositi is a double heterozygote for Hb 0 Ar/beta(0)-thalassemia. His father is a simple heterozygote for Hb 0 Ar with clear-cut cytomorphological stigmata, indicating hemoglobinosis. The second propositus, according to clinical and laboratory data is also a double heterozygote for Hb 0 Ar and beta(0)-thalassemia. The carriers investigated are of Bulgarian nationality. Their territorial origin supportsthe thesis that the gene mutation for Hb 0 Arab most probably has taken place out of the present boundaries of Bulgaria.     

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.     

Heme structures of five variants of hemoglobin M probed by resonance Raman spectroscopy

The alpha-abnormal hemoglobin (Hb) M variants show physiological properties different from the beta-abnormal Hb M variants, that is, extremely low oxygen affinity of the normal subunit and extraordinary resistance to both enzymatic and chemical reduction of the abnormal met-subunit. To get insight into the contribution of heme structures to these differences among Hb M's, we examined the 406.7-nm excited resonance Raman (RR) spectra of five Hb M's in the frequency region from 1700 to 200 cm(-1). In the high-frequency region, profound differences between met-alpha and met-beta abnormal subunits were observed for the in-plane skeletal modes (the nu(C=C), nu(37), nu(2), nu(11), and nu(38) bands), probably reflecting different distortions of heme structure caused by the out-of-plane displacement of the heme iron due to tyrosine coordination. Below 900 cm(-1), Hb M Iwate [alpha(F8)His --> Tyr] exhibited a distinct spectral pattern for nu(15), gamma(11), delta(C(beta)C(a)C(b))(2,4), and delta(C(beta)C(c)C(d))(6,7) compared to that of Hb M Boston [alpha(E7)His --> Tyr], although both heme irons are coordinated by Tyr. The beta-abnormal Hb M variants, namely, Hb M Hyde Park [beta(F8)His --> Tyr], Hb M Saskatoon [beta(E7)His --> Tyr], and Hb M Milwaukee [beta(E11)Val --> Glu], displayed RR band patterns similar to that of metHb A, but with some minor individual differences. The RR bands characteristic of the met-subunits of Hb M's totally disappeared by chemical reduction, and the ferrous heme of abnormal subunits was no longer bonded with Tyr or Glu. They were bonded to the distal (E7) or proximal (F8) His, and this was confirmed by the presence of the nu(Fe-His) mode at 215 cm(-1) in the 441.6-nm excited RR spectra. A possible involvement of heme distortion in differences of reducibility of abnormal subunits and oxygen affinity of normal subunits is discussed.     

Hemoglobin Roseau-Pointe a Pitre alpha 2 beta 2(90) (F6) Glu----Gly: a new hemoglobin variant with slight instability and low oxygen affinity

A Dominican neonate carrying a new abnormal hemoglobin, hemoglobin Roseau Pointe-à-Pitre alpha 2 beta 2(90)(F6) Glu----Gly, was detected in Guadeloupe during application of a cord blood screening program. This variant behaved in isoelectrofocusing as an Hb D, and displayed instability and low whole blood oxygen affinity. In the affected family it was present, either isolated, or in association with a beta+ thalassemia trait.     

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) .     

Tertiary structural analysis of the elongated part of an abnormal hemoglobin, hemoglobin Pakse

Hemoglobin variants in which a frameshift results in chain elongation are unusual. Hemoglobin Pakse (Hb Pakse) is an unstable hemoglobin with abnormal elongation, first described in Indochina. An alpha2-globin gene termination codon mutation, TAA -->TAT or Term -->Tyr, has been described in the pathogenesis of Hb Pakse. This abnormality causes a frameshift that elongates the alpha chain amino acids. Computer-based protein structure modeling was used in a bioinformatics analysis of the tertiary structure of these elongated amino acid sequences. The elongated part of Hb Pakse showed additional helices, which may cause the main alteration in Hb Pakse. Abnormalities in the fold structure of globin in Hb Pakse were identified, and helices additional to the normal alpha globin chains were shown in the elongated part of Hb Pakse.     

Location of amino acid substitutions in human hemoglobin. Mass spectrometric rapid analysis of tryptic peptides

Point mutations alpha 58 His----Tyr (Hb M Boston), beta 6 Glu--lys (Hb C) and beta 26 Glu----Lys (Hb E) have been identified in abnormal hemoglobins by means of tryptic hydrolysis of their alpha- and beta-chains followed by mass-spectrometry coupled with direct extraction of ions from solution. The abnormal hemoglobin Hb M Boston alpha 58 (E7) His----Tyr has been for the first time found in the blood of a patient from the USSR. This express-method is generally applicable for the identification of point mutation in proteins. The amount of protein necessary for the analysis is 100-1000 pmole. The stability, proteolytic degradation of the identified abnormal Hb's and Hb Bart's were investigated. The molecular pathogenesis of the hemoglobinopathies are discussed from the point of view of the observed properties.     

Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 beta and 145 beta mutations.

In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.     

Hemoglobin Tilburg: alpha 2-beta 2 73 (E 17) Asp----Gly. A new hemoglobin with reduced oxygen affinity

A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the beta-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp----Asn), Hb Mobile (Asp----Val) and Hb Vancouver (Asp----Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at beta 73.     

Human hemoglobin Portland II (zeta 2 beta 2). Isolation and characterization of Portland hemoglobin components and their constituent globin chains

Two types of embryonic hemoglobins (Hb) containing zeta chains have been identified in the blood of several neonates of Chinese origin with homozygous alpha-thalassemia. In addition to Hb Portland I (zeta 2 gamma 2) which was previously reported, another embryonic hemoglobin has been detected and found to contain zeta chains and beta chains. It is being designated Hb Portland II and has the formula (zeta 2 beta 2). It has a mobility slightly slower than that of Hb A on starch gel electrophoresis at pH 8.6 and has been found in the hemolysates of blood of some but not all hydropic infants. Another component with a mobility faster than that of Hb A2 on starch gel has been isolated from the blood of some hydropic neonates. This latter component is postulated to be zeta 2 delta 2. The occurrence of Hb Portland I and Hb Portland II in these hydropic neonates is consistent with the hypothesis that, in the absence of normal alpha chain production, zeta chains are continued to be produced at later states of development than normal and form tetramers with each of the beta-like globin chains. Because Hb Portland II has not been found in blood from all hydropic neonates, we postulate that the presence of this hemoglobin in these fetuses may be correlated with the gestational age of the fetus at the time of birth.     

Hemoglobin Kariya [alpha 40 (C5) Lys leads to Glu]: a new hemoglobin variant with an increased oxygen affinity

A new abnormal hemoglobin, Hb Kariya [alpha 40 (C5) Lys leads to Glu], with an amino acid substitution at the alpha 1 beta 2 contact was discovered in a young Japanese man. This variant migrated to the anode faster than Hb A, being nearly the same as Hb I in electrophoretic mobility. It amounted to about 6% of the total hemoglobin of the hemolysate. This hemoglobin showed an increased oxygen affinity, decreased heme-heme interaction and a lowered 2,3-DPG effect.     

Structure of deoxy hemoglobin C (beta six Glu replaced by Lys) in two crystal forms

Five crystal forms of the abnormal human hemoglobin Hb³ C (beta six Glu → Lys) have been grown. Two of them are grown with liganded Hb C, three with deoxy Hb C. The structures of two of the deoxy crystal forms were determined by the method of molecular replacement, using deoxy Hb A as the model structure. Fourier maps were calculated for each Hb C structure, using data to a resolution of 5 Å in one case and 4 Å in the second case. The structural differences between each deoxy Hb C structure and the deoxy Hb A model are found mostly at the molecular surface. Energetically favorable interactions involving the variant residue, beta six lysine, occur in both Hb C crystal forms, and could explain the lowered solubility and enhanced tendency of deoxy Hb C to crystallize in vivo.     

Structure-function relationship in a variant hemoglobin: a combined computational-experimental approach

Our study examines the functional and structural effects of amino acid substitution in the distal side of beta-chains of human Hb Duarte (alpha(2)beta(2)(62Ala-->Pro)). We have compared the functional properties of the purified Hb Duarte with those of HbA, and through proton NMR and molecular dynamics simulations we have investigated their tertiary and quaternary structures. The variant exhibits an increased oxygen affinity with a normal Hill coefficient and Bohr effect. The abnormal function of Hb Duarte is attributed to the presence of a proline residue at the beta62 position, since the functional properties of another Hb variant in the same position, Hb J-Europa (beta(62Ala-->Asp)), have been described as normal. Thereafter (1)H-NMR studies have shown that the beta62 Ala-->Pro substitution causes structural modifications of the tertiary structure of the beta globins, leaving the quaternary structure unaltered. These results have been confirmed by extensive all-atom molecular dynamics simulations. All these findings lead to the conclusion that the beta62 Ala-->Pro substitution produces a destabilization of the E-helix extending downward to the CD corner. Particularly, a cavity near the distal histidine of the beta-chains, connecting the heme pocket to the solvent, is affected, altering the functional properties of the protein molecule.