Polymerization and Solubility of Recombinant Hemoglobins α2β26VAL(HB S) and α2β26LEU(HB LEU)

Adachi, K.; Rappaport, Eric; Eck, H S; Konitzer, P; Kim, J; Surrey, Saul

doi:10.3109/03630269108998861

Cited by 16 publications

(33 citation statements)

References 19 publications

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“…The synthetic human a-and 3-globin genes for normal Hb (17) and for a few other mutant Hbs (18)(19)(20)(21)(22)27) have been expressed in E. coli. However, the Hb produced in this system is not processed at its N terminus in the same way that human Hb is processed in human reticulocytes.…”

Section: Discussionmentioning

confidence: 99%

Recombinant human sickle hemoglobin expressed in yeast.

Llano

Schneewind

Stetler

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Sickle hemoglobin has been expressed in the yeast Saceharomyces cerevwisa after site-diected mutagenesis of a plasmid conti normal human a-and I3-globin genes.Cassette mutagenesis of this plasnid was achieved by inserting a DNA fragment containing the 3-globin gene in the replicative form of M13mpl8 to make a point mutation and then recondfIy the original paid containing the mutated -globin gene. Pure recombinant hemolbin S was shown to be identical to natural sickle hemoglobin in its ultraviolet and visible absorption bands and by gel dectrophoresi, iboectric focusing, amino acid analysis, mass spectrometry, partial N-terminal sequencing, and finctional properties (Pso, cooperativity, and response to 2,3-bisphosphoglycerate). In yeast and in mammalian cells, cotranlational processing yields the same N-terminal valine residues of hemoglobin a-and a-chains, but in bacterial expression systems the N terminus Is extended by an additional amino acid because the initiator methionine residue is retained. Since the N-terminal valine residues of both chains of hemoglobin S participate in important physiological hunctions, such as oxygen affinity, interaction with anions, and the Bohr coefficient, the yeast expression system is preferable to the bacterial system for recombinant DNA studies. Hence, mutagenesis employing this expression system should permit definitive as ets of the role of any amino acid side chain in hemoglobin S aggregation and could suggest additional approaches to therapeutic intervention. The engineering ofthis system for the synthesis of sickle hemoglobin and its purification to homogeneity in a single column procedure are described.The genetic mutation underlying sickle cell anemia leads to the replacement of Glu-6 on the (-chain with a valine residue (1, 2). The substitution of a hydrophobic side chain for a hydrophilic one on the exterior ofthe protein (3) promotes the aggregation of deoxygenated hemoglobin (Hb) tetramers (4, 5) that eventually distort the erythrocyte into a sickled form in the venous circulation (6). One approach toward treatment of sickle cell anemia focuses on the HbS molecule itself by reacting it with chemical modifiers that directly alter the functional properties of the protein and indirectly reduce aggregation in vitro (7,8). The number and nature of such susceptible sites has been limited to those hydrophilic side chains with enhanced reactivity because of their location in the protein (8, 9). The availability of recombinant DNA technology now permits studies at any site on the HbS tetramer, such as hydrophobic amino acid side chains heretofore not possible to modify by other methods. For sickle cell HbS, the ability to alter hydrophobic sites is especially important since the initial and some of the subsequent stages of aggregation involve hydrophobic interactions. Although the identity of some of these contact sites in the aggregate is known (3)(4)(5)10), there is a lack of information on other contact sites and their contribution to the overall strength of the ...

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Section: Discussionmentioning

confidence: 99%

Recombinant human sickle hemoglobin expressed in yeast.

Llano

Schneewind

Stetler

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Cells from overnight growths at 30°C were lysed and treated with DNase I, and soluble proteins were removed by centnfugation [18]. After 3-5 washes with 0.5% (v/v) Triton X-100, the resultant pellet was dissolved in 8 M urea/SO mM Tris buffer and the fusion protein was purified by ion-exchange chromatography (CM-Sepharose).…”

Section: Methodsmentioning

confidence: 99%

“…These results indicate that polymerization is not specific for valine at the j36 position, and that an abrupt decrease in deoxyhemoglobin solubility may depend, in general, on hydrophobic amino acids at the 86 position. To further understand the relationship between 86 amino acid hydrophobicity and deoxyhemoglobin solubility, we compared solubilities in high phosphate buffers for #J6 Trp and Ala hemoglobins with those of 86 Glu (Hb A), Val (Hb S), Leu (aAL""), Ile (cx&"~) and Gln (Hb Machida) [17,18,21] (Table I).…”

Section: Solubility Of Hemoglobins Modijied At the 86 Positionmentioning

confidence: 99%

Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

et al. 1993

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The relationship between different amino acids at the/l6 position of hemoglobin and tetramer stability was addressed by a site-directed mutagenesis approach. Precipitation rates during mechanical agitation of oxyhemoglobins with Gln, Ala, Val, Leu and Trp at the 86 position increased 2, 5, 13, 21 and 53 times, respectively, compared with that for Hb A. There was a linear relationship between the log of the precipitation rate constant and amino acid hydrophobicity at the 86 position, suggesting that enhanced precipitation of oxy Hb S during mechanical agitation results in part from increased hydrophobic&y of/?6 Val. Deoxyhemoglobin solubility increased in the order of/36 Be, Leu, Val, Trp, Gln, Ala and Glu suggesting that hydrophobic interactions between 86 Val and the acceptor site of another hemoglobin molecule during deoxy-Hb S polymerization not only depend on hydrophobicity but also on stereospecificity of the amino acid side chain at the 86 position. Furthermore, our results indicate that hydrophobic amino acids at the 86 position which promote tetramer instability in the oxy form do not necessarily promote polymerization in the deoxy form.

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“…We previously engineered and isolated Hb S ␤T87Q as well as Hb F ␥E6V and Hb F ␥E6V,␥Q87T, using a yeast expression system, and characterized the polymerization properties of these variants to clarify the role of Gln at ␥87 in Hb F-mediated inhibition of deoxy Hb S polymerization (12,13). Oversaturated deoxy Hb F ␥E6V and Hb F ␥E6V,␥Q87T polymerized similarly without a delay time at a higher concentration than Hb S, even though studies of mixtures of Hb S and Hb S ␤T87Q indicated that the Thr 3 Gln difference at 87 (F3) in the non-␣-globin chain is a key amino acid required for Hb F inhibition of Hb S polymerization.…”

Section: From the Children's Hospital Of Philadelphia Division Of Hementioning

confidence: 99%

Polymerization of Three Hemoglobin A2 Variants Containing Valδ6 and Inhibition of Hemoglobin S Polymerization by Hemoglobin A2

Adachi

Pang

Reddy

et al. 1996

Journal of Biological Chemistry

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To understand determinants for hemoglobin (Hb) stability and Hb A 2 inhibition of Hb S polymerization, three Val ␦6 Hb A 2 variants (Hb A 2 ␦E6V, Hb A 2 ␦E6V,␦Q87T, and Hb A 2 ␦E6V,␦A22E,␦Q87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed. Oxy forms of Hb A 2 ␦E6V and Hb A 2 ␦E6V,␦Q87T were 2-and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A 2 ␦E6V,␦A22E,␦Q87T was similar to that of Hb S, suggesting that Ala ␦22 and Gln ␦87 contribute to the surface hydrophobicity of Hb A 2 . Deoxy Hb A 2 ␦E6V polymerized without a delay time, like deoxy Hb F ␥E6V, while deoxy Hb A 2 ␦E6V,␦Q87T and deoxy Hb A 2 ␦E6V,␦A22E,␦Q87T polymerized after a delay time, like deoxy Hb S, suggesting that ␤87 Thr is required for the formation of nuclei. Deoxy Hb F ␥E6V,␥Q87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization, suggesting that Thr effects on Val

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Polymerization and Solubility of Recombinant Hemoglobins α₂β₂^6VAL(HB S) and α₂β₂^6LEU(HB LEU)

Cited by 16 publications

References 19 publications

Recombinant human sickle hemoglobin expressed in yeast.

Recombinant human sickle hemoglobin expressed in yeast.

Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

Polymerization of Three Hemoglobin A2 Variants Containing Valδ6 and Inhibition of Hemoglobin S Polymerization by Hemoglobin A2

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