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• W2079109460 abstract "Authentic soluble human β-globin chains were produced in Escherichia coli using an expression plasmid (pHE2β) containing full-length cDNAs coding for human β-globin chain and methionine aminopeptidase. Spectral properties of the purified β-globin were identical to those of authentic β-globin. Soluble β-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric β-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric β-globin. Monomeric forms of β-globin, like authentic native β-globin, formed tetrameric hemoglobin (Hb) A (α2β2) in vitro upon incubation with α-globin, while dimeric forms did not. When β-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the β-globin chain monomers assembled with α-globin and formed hemoglobin tetramers. α-Globin was more thermally unstable than β-globin, while assembled tetramers promoted higher stability. Disulfide-bonded β-globin dimers showed a slight increase in thermal stability compared with β-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of α chains favors assembly with β-globin, β-β dimers cannot bind α chains, and that Hb A tetramer formation results in the most thermally stable species. Authentic soluble human β-globin chains were produced in Escherichia coli using an expression plasmid (pHE2β) containing full-length cDNAs coding for human β-globin chain and methionine aminopeptidase. Spectral properties of the purified β-globin were identical to those of authentic β-globin. Soluble β-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric β-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric β-globin. Monomeric forms of β-globin, like authentic native β-globin, formed tetrameric hemoglobin (Hb) A (α2β2) in vitro upon incubation with α-globin, while dimeric forms did not. When β-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the β-globin chain monomers assembled with α-globin and formed hemoglobin tetramers. α-Globin was more thermally unstable than β-globin, while assembled tetramers promoted higher stability. Disulfide-bonded β-globin dimers showed a slight increase in thermal stability compared with β-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of α chains favors assembly with β-globin, β-β dimers cannot bind α chains, and that Hb A tetramer formation results in the most thermally stable species. The development of molecular biological techniques to replace selectively individual amino acids has helped further our understanding of the relationship between structure and function of hemoglobin. Initial reports described production of normal and modified human β-globin in bacteria employing a fusion protein expression vector (1Nagai K. Thogerson H.C. Nature. 1984; 309: 810-812Crossref PubMed Scopus (323) Google Scholar). An expression system was later developed in which α- and β-globin chains were coexpressed, resulting in formation of soluble tetrameric hemoglobin in yeast (2Wagenbach M. O'Rourke K. Vitez L. Wieczorek A. Hoffman S. Durfee S. Tedesco J. Stetler G. Bio/Technology. 1991; 9: 57-61Crossref PubMed Scopus (111) Google Scholar, 3Adachi K. Konitzer P. Lai C.H. Kim J. Surrey S. Protein Eng. 1992; 5: 807-810Crossref PubMed Scopus (33) Google Scholar). Recent studies described coexpression of human α- and β-globin in Escherichia coli, which resulted in formation of soluble tetrameric hemoglobins (4Hoffman S.J. Looker D.L. Roehrich J.M. Cozart P.E. Durfee S.L. Tedesco J.L. Stetler G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8521-8525Crossref PubMed Scopus (220) Google Scholar), as well as a system for high expression of insoluble β-globin chains in E. coli. (5Hernan R.A. Hui H.L. Andracki M.E. Noble R.W. Sligar S.G. Walder J.A. Walder R.Y. Biochemistry. 1992; 31: 8619-8628Crossref PubMed Scopus (74) Google Scholar). These last two systems, however, result in globin chains containing N-terminal methionine, which may affect functional properties of hemoglobin. More recently, Shen et al. (6Shen T-J. Ho N.T. Simplaceanu V. Zou M. Green B.N. Tam M.F. Ho C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8108-8112Crossref PubMed Scopus (138) Google Scholar) expressed soluble hemoglobin tetramers lacking N-terminal methionine in bacteria by coexpression of α- and β-globin cDNAs with methionine aminopeptidase cDNA. In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4Hoffman S.J. Looker D.L. Roehrich J.M. Cozart P.E. Durfee S.L. Tedesco J.L. Stetler G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8521-8525Crossref PubMed Scopus (220) Google Scholar, 5Hernan R.A. Hui H.L. Andracki M.E. Noble R.W. Sligar S.G. Walder J.A. Walder R.Y. Biochemistry. 1992; 31: 8619-8628Crossref PubMed Scopus (74) Google Scholar, 7Groebe D.R. Busch M.R. Tsao T.Y.M. Luh F.Y. Tam M.F. Chung A.E. Gaskell M. Liebhaber S.A. Ho C. Protein Expression Purif. 1992; 3: 134-141Crossref PubMed Scopus (9) Google Scholar, 8Fronticelli C. O'Donnell J.K. Brinigar W.S. J. Protein Chem. 1991; 10: 495-501Crossref PubMed Scopus (30) Google Scholar). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization. After solubilization, chains must then be renatured in vitro and form correctly folded native globin chains, which then must properly assemble to form authentic hemoglobin tetramers (1Nagai K. Thogerson H.C. Nature. 1984; 309: 810-812Crossref PubMed Scopus (323) Google Scholar, 7Groebe D.R. Busch M.R. Tsao T.Y.M. Luh F.Y. Tam M.F. Chung A.E. Gaskell M. Liebhaber S.A. Ho C. Protein Expression Purif. 1992; 3: 134-141Crossref PubMed Scopus (9) Google Scholar, 8Fronticelli C. O'Donnell J.K. Brinigar W.S. J. Protein Chem. 1991; 10: 495-501Crossref PubMed Scopus (30) Google Scholar, 9Nagai K. Thogerson H.S. Methods Enzymol. 1987; 153: 461-481Crossref PubMed Scopus (344) Google Scholar). This process is labor intensive, and efficiency of tetramer reconstitution from denatured globin chains is very low. These limitations have made it impractical to produce efficiently modified soluble authentic β-globin chains in order to study assembly in vitro with α chains. In this report, we describe expression of soluble authentic β-globin employing an E. coli expression system that contains cDNAs for human β-globin and methionine aminopeptidase. In addition, we describe efficient assembly in vitro of purified β- with α-globin to form hemoglobin tetramers and, furthermore, report results on effects of β-β dimerization on thermal stability and assembly in vitro with α-globin. The original pHE2 plasmid (kindly provided by Drs. C. Ho and T-J. Shen, Carnegie Mellon University, Pittsburgh, PA) was constructed to coexpress α- and β-globin chains with methionine aminopeptidase (10Ben-Bassat A. Bauer K. Chang S.-Y. Myambo K. Boosman A. Chang S. J. Bacteriol. 1987; 169: 751-757Crossref PubMed Google Scholar) under transcriptional control of a tac promoter in order to obtain soluble authentic human Hb A without N-terminal methionine (6Shen T-J. Ho N.T. Simplaceanu V. Zou M. Green B.N. Tam M.F. Ho C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8108-8112Crossref PubMed Scopus (138) Google Scholar). In order to achieve expression of authentic β-globin chain alone, the α-globin cDNA was removed from pHE2 by digestion with XbaI (Life Technologies, Inc.), the 6.3-kilobase pair fragment, which contains β-globin and methionine aminopeptidase cDNAs, was purified using a Geneclean II kit (BIO 101, Inc.) and ligated by incubation with T4 DNA ligase (Life Technologies, Inc.). A diagram of the β-globin-expressing plasmid pHE2β is shown in Fig. 1. The plasmid was transfected into E. coli (JM 109) (Promega Co., Madison, WI), and bacteria were grown at 30°C with shaking at 225 rpm in 6 liters of TB (terrific broth) containing 100 μM ampicillin to a density of about 3 × 1010 bacteria/ml. β-Globin chain expression was induced for 6 h at 30°C by addition of 0.2 mM isopropyl-β-D-thiogalactopyranoside (Fisher Scientific, Fair Lawn, NJ), and cultures were then supplemented with 10 μM hemin (Aldrich) and 0.1% (w/v) glucose. After the 6-h incubation, bacterial cultures were saturated with CO gas to convert expressed β-globin to the CO form. Bacteria were pelleted by centrifugation at 2,300 × g for 10 min, resuspended in 10 mM phosphate buffer, pH 8.6, lysed by sonication at 4°C, and then centrifuged at 4°C for 45 min at 27,000 × g. Soluble β-globin chains were purified as described below at 4°C, and CO gas was introduced at each purification step to maintain β-globin in the CO form. The supernatant was applied to a DEAE-cellulose (Sigma) column equilibrated with 10 mM phosphate buffer, pH 8.6, the column was washed with 5 column volumes of buffer, and then fractions were eluted with 50 mM phosphate buffer, pH 6.3. The partially purified β-globin fraction was rechromatographed on a Mono Q column equilibrated with 10 mM phosphate buffer, pH 8.6, and was further purified with a linear gradient from 10 mM phosphate buffer, pH 8.6, to 50 mM phosphate buffer, pH 6.3. The purified soluble β-globin was then concentrated by Centriprep 10 (Amicon Inc., Beverly, MA) and stored at −70°C. Separation of polymeric and monomeric forms of β-globin was achieved by gel filtration on a Superose 12 column in 100 mM potassium phosphate buffer, pH 7.0. Authentic human α- and β-globin chains were purified from tetrameric Hb A isolated from erythrocyte lysates according to previously described methods (11Ikeda-Saito M. Inubushi T. Yonetani T. Methods Enzymol. 1981; 76: 113-121Crossref PubMed Scopus (20) Google Scholar). Removal of p-mercuribenzoate was accomplished using 20 mM DTT, 1The abbreviations used are: DTTdithiothreitolHbhemoglobinHPLChigh performance liquid chromatographyES-MSelectrospray mass spectrometryPAGEpolyacrylamide gel electrophoresis. and globin chains were isolated by gel filtration on a Superose 12 column. dithiothreitol hemoglobin high performance liquid chromatography electrospray mass spectrometry polyacrylamide gel electrophoresis. Molecular mass and sample purity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described (12Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (204097) Google Scholar). In addition, mass determination was done to confirm independently results from SDS-PAGE and to evaluate N-terminal methionine cleavage from β-globin chains. Electrospray ionization mass spectrometry (ES-MS) was performed on a VG BioQ triple quadrupole mass spectrometer (Micromass, Altrincham, Cheshire, United Kingdom) (13Shackleton C.H. Witkowska H.E. Desiderio D.M. Mass Spectrometry: Clinical and Biomedical Applications. Vol 2. Plenum Press, New York1994: 135Google Scholar). The multiply charged ions derived from α-globin (Mr 15,126.4) served as an internal or external standard for mass scale calibration when analyzing Hb A and β-globin proteins, respectively. MS/MS experiments were performed on peptides collected in the course of a narrow bore, reversed-phase HPLC/ESMS analysis of the tryptic digest derived from DTT-treated fraction 1 (Fig. 2). Doubly charged molecular ions at 1030 and 1038 m/z corresponding to βT5 and its oxidized form, respectively, were utilized as processor ions in the process of collision-induced dissociation. Argon pressure was 10−3 mbar, collision energy was 25 V, and 5.9-s MS-2 scans were acquired between 180 and 2100 m/z in continuum mode. Data analysis employed the MassLynx® software package (Micromass, Altrincham, Cheshire, UK). Globin samples were also precipitated with acid (HCl) acetone (14Ascoli F. Rossi-Fanelli M.R. Antonini E. Methods Enzymol. 1981; 76: 72-87Crossref PubMed Scopus (227) Google Scholar), dissolved in 6 M guanidine/ammonium bicarbonate buffer, and subjected to tryptic digestion. Resulting peptides were analyzed by narrow bore, reversed-phase HPLC/ES-MS (15Schroeder W.A. Hancock W.S. CRC Handbook of HPLC for the Separation of Amino Acids, Peptides, and Proteins. Vol II. CRC Press, Boca Raton, FL1984: 287Google Scholar, 16Witkowska H.E. Bitsch F. Shackleton C.H.L. Hemoglobin. 1993; 17: 227-242Crossref PubMed Scopus (52) Google Scholar). Purified β-globin chains were also analyzed by cellulose acetate electrophoresis, and mobilities were compared with those of authentic human globin chains. Analysis of hemoglobin tetramer formation in vitro using purified β-globin and α-globin prepared from human red blood cells was done using cellulose acetate electrophoresis on Titan III membranes at pH 8.6 with Super-Heme buffer (Helena Laboratories, Beaumont, TX). Absorption spectra of the purified β-globin in the CO form was recorded using a Hitachi U-2000 spectrophotometer. Circular dichroism (CD) spectra of hemoglobins were recorded using an Aviv-model 62DS instrument employing a 0.1-cm light path cuvette at ∼10 μM hemoglobin concentrations, and temperature was controlled with a thermoelectric module. Thermal stability of globin chains in 0.1 M phosphate buffer, pH 7.0, was evaluated by monitoring temperature-induced changes in ellipticity at 222 nm as a function of time at 60°C. β-Globin chains in the CO form (5 μg) purified from human red blood cells were incubated in the presence or absence of 1 unit of bovine liver catalase (Sigma) and then assessed for β-β dimer formation. β-Globin was also incubated for 0.5 h at 25°C in the presence of various concentrations of hydrogen peroxide (Sigma), and dimer formation was quantitated by SDS-PAGE using a densitometer (Helena Laboratories). Soluble human β-globin was expressed in E. coli and purified by a combination of two anion-exchange chromatography steps using DEAE-cellulose and Mono Q. Approximate yield of soluble purified β-globin was 2-3 mg/liter culture, which represents about 40% of the yield when β chains are coexpressed with α chains to form soluble tetrameric Hb A using the same conditions. The β-globin fraction eluted as a single peak from Mono Q chromatography; however, SDS-PAGE showed two major bands of 16 and 32 kDa (data not shown). Further purification of the single peak from Mono Q chromatography using Superose 12 gel filtration chromatography showed at least two components (Fig. 2). SDS-PAGE of fraction 1 (Fr. 1) showed a diffuse doublet with molecular mass corresponding to about 32 kDa (Fig. 3). Presence of the higher molecular mass species of the doublet near 32 kDa was not consistent and varied depending on batch. On the other hand, fraction 2 (Fr. 2) migrated as a single band on SDS-PAGE with a molecular mass of about 16 kDa, which corresponds to that of authentic human β-globin chain. Treatment of fraction 1 with 50 mM DTT for 1 h resulted in complete conversion of the 32-kDa to a 16-kDa form, which comigrated on SDS-PAGE with the 16-kDa band of fraction 2; DTT treatment of fraction 2 had no effect on band migration on SDS-PAGE. These results suggest that fraction 1 contains β-globin homodimers linked by an intermolecular disulfide bond, while fraction 2 corresponds to monomeric β-globin. Electrospray mass spectrometry of fraction 1 revealed two species: a major one of 31,733.8 Da and a minor one of 31,768.4 Da (Fig. 4, top). Molecular mass of the major species corresponds to the expected mass of a disulfide-bonded human β-globin dimer (Mr 31,732.4). The minor species shows a 34.6-Da mass increment over the major component, consistent with addition of two oxygen atoms per β-β-globin chain. The minor species may correspond to the higher molecular weight band in the doublet observed in SDS-PAGE. Addition of DTT to fraction 1 resulted in conversion of both dimeric species to their monomeric counterparts. SDS-PAGE revealed a single band at ∼16 kDa, while ESMS analysis detected two species. As illustrated in Fig. 4, lower panel, DTT-treated fraction 1 contains a major component of 15,867.1 Da that corresponds to βA-globin (Mr 15,867.2) and a minor component (15,886.5 Da), which is consistent with an addition of one oxygen atom to β-globin. This result also indicates complete cleavage of the N-terminal, chain-initiating methionine from β-globin in bacteria by the coexpressed methionine aminopeptidase. The normal human β-globin chain contains two cysteine residues at β93 and at β112, within tryptic peptides βT10 (β83-95) and βT12 (β105-120), respectively. In order to identify which cysteine was involved in disulfide bond formation during protein expression and/or purification, monomeric and dimeric β-globin fractions were subjected to proteolytic digestion with trypsin, and peptides were separated and identified by HPLC/ES-MS analysis. In the digests derived from both β-β dimer and its DTT-reduced monomeric form, the following cysteine-containing peptides were observed, all at low levels: βT10, βT10-βT10 and βT10-βT12. At the same time, peptides βT12 and βT12-βT12 (encompassing Cysβ112) were not detected in any of the digests. Presence of βT10-βT10 and βT10-βT12 peptides in a digest derived from the DTT-reduced globin implies that these disulfide-bonded species are products of nonspecific oxidation, likely occurring during proteolytic digestion and/or handling of peptide mixtures; this phenomenon was noted previously by others (17Nakanishi T. Kishikawa M. Shimizu A. Hayashi A. Inoue F. J. Mass Spectrom. 1995; 30: 1663-1670Crossref Scopus (14) Google Scholar). In addition, no difference in relative abundance of βT10-βT10 and βT10-βT12 was seen when comparing the tryptic digests derived from β-globin dimer and monomer. It can then be inferred that neither βT10-βT10 nor βT10-βT12 represents a major dimerization site in the β-β-globin. Thus, since the above results argue against Cysβ93 being a primary site of an intermolecular disulfide bond formation, it follows that Cysβ112 must be involved in the dimerization event. So far, we were unable to unequivocally prove this due to difficulties in handling and detection of the core βT12 peptide (15Schroeder W.A. Hancock W.S. CRC Handbook of HPLC for the Separation of Amino Acids, Peptides, and Proteins. Vol II. CRC Press, Boca Raton, FL1984: 287Google Scholar, 16Witkowska H.E. Bitsch F. Shackleton C.H.L. Hemoglobin. 1993; 17: 227-242Crossref PubMed Scopus (52) Google Scholar). Experiments are now in progress to mutagenize Cysβ112 to evaluate effects on dimer formation. While peptide mapping left some ambiguity regarding the site of dimerization, it confirmed the faithfulness of β-globin expression and provided evidence regarding the nature of a 34.6-Da addition to the β-globin dimer. Specifically, complete cleavage of the N-terminal methionine was substantiated by detection of an authentic N-terminal βT1 peptide, while the putative extended Met-βT1 was absent in the digest. In accord with detection of putative oxygenated β-globin species (Fig. 4, top and bottom), a peptide that measured 16 Da more than βT5 (β41-59) was found in tryptic digests derived from the dimeric and monomeric components of fraction 1. Sequencing of this peptide by MS/MS on a triple quadrapole electrospray mass spectrometer (data not shown) narrowed down the position of this modification to the last five amino acid residues (Met55-Lys59). A single methionine residue in β-globin is located within the above sequence (Met55), and thus it's oxidation to methionine sulfoxide seems a likely explanation for the observed increment in peptide (and globin) mass. The purified soluble β-globin chains (CO form) showed a typical absorption spectra characteristic of human hemoglobin (18Di Iorio E.E. Methods Enzymol. 1981; 76: 57-72Crossref PubMed Scopus (239) Google Scholar) with peaks at 568, 540, 419, 344, and 276 nm. The ratio of absorbance at 540 nm to that at 276 nm was about 0.3, was consistent among different batches, and was similar to the corresponding value for native, authentic human β-globin. Tetramer assembly in vitro was evaluated for each of the two purified fractions from the Superose 12 column after addition of authentic human native α-globin chain obtained from human red blood cells (Fig. 5). Under these conditions (e.g. 10 mM potassium phosphate buffer, pH 7.0 at 25°C) authentic, native human β-globin chain very rapidly formed Hb A tetramers (α2β2) in vitro with α-globin (Fig. 5, lane 4). Fraction 2, which contains monomeric β-globin chains, also rapidly formed Hb A tetramers with α-globin chains (Fig. 5, lanes 5 and 6). Formation of tetramers depended on total amount of α- and β-globin chains. If there were an excess of β chains, all of the added α chains were converted with an equivalent amount of β chains to tetramers; in contrast, fraction 1, which contains β-β dimers, was unable to form tetramers in the presence of α chains, even when the same amount of β chain present in fraction 2 was mixed with α chains (Fig. 5A, lane 7). Hb A tetramer formation was not observed on cellulose acetate electrophoresis even after doubling the amount of fraction 1, which was incubated with the α-globin chains (Fig. 5, lane 8). If, however, fraction 1 were first treated with 20 mM DTT for 1 h at 25°C to convert β-β dimers to monomers, then tetrameric Hb A readily formed upon addition of α-globin chains (Fig. 5, lanes 9 and 10). These results indicate that intermolecular disulfide bond formation between two β-globin chains results in β-β dimers, which inhibit assembly with α chains and results in no assembly of tetrameric Hb A. Purified native human β-globin chains from red blood cells can undergo dimerization during storage or under conditions favoring oxidation, and these dimers are also not productive in assembly with α-globin chains. Spontaneous conversion of monomeric β-globin chains prepared from human red blood cells to disulfide-bonded dimers was evident during storage and was promoted by incubation at higher temperatures (Fig. 6A). Dimer formation was observed after 0.5 h incubation at 37°C, and the amounts increased as a function of time. Furthermore, after a 3-h incubation, small amounts (∼10%) of higher molecular mass products (>46 kDa) were also observed. In contrast, addition of catalase during incubation inhibited dimer formation (Fig. 6A). Incubation of monomeric β-globin chains with hydrogen peroxide also promoted dimer formation and the amounts increased with increasing peroxide concentration (Fig. 6B). It is generally known that individual α- or β-globin chains are more unstable than tetrameric Hb A and that α-globin is more unstable than β-globin (19Asakura T. Adachi K. Sono M. Friedman S. Schwartz E. Biochem. Biophys. Res. Commun. 1974; 57: 780-786Crossref PubMed Scopus (20) Google Scholar, 20Friend S.H. Matthew J.B. Gurd F.R.N. Biochemistry. 1981; 20: 580-586Crossref PubMed Scopus (23) Google Scholar). Furthermore, proper disulfide bond formation during folding generally promotes protein stability (21Baldwin R.L. Eisenberg D. Oxender D.L. Fox C.F. Protein Engineering. Alan R. Liss, Inc., New York1987: 127Google Scholar, 22Klibanov A.M. Ahern T.J. Oxender D.L. Fox C.F. Protein Engineering. Alan R. Liss, Inc., New York1987: 213Google Scholar). Thermostability of purified monomeric α- and β-globins as well as dimeric β-globin in the CO forms was measured in 0.1 M phosphate buffer, pH 7.0 at 60°C, by monitoring temperature-induced CD changes in ellipticity at 222 nm. CD spectrum for Hb A and α-, β-, and β-β-globin dimers were similar at low temperature (10°C) in the region from 190 to 260 nm (Fig. 7). Changes in ellipticity at 222 nm for Hb A and α-, β-, and β-β-globin dimers as a function of time after temperature jump to 60°C are shown in Fig. 8. Changes in the CD spectra under these experimental conditions follow first-order kinetics. Changes were more dramatic for α-globin than for Hb A tetramers and plateaued after about 1,000 s, suggesting rapid temperature-induced unfolding of α-globin chain. The initial rate of change for β-globin was more than that for Hb A, but much less than that for α-globin, while that for β-β-globin dimers was slightly less than that for unmodified β-globins. Results of thermal denaturation for Hb A, dimeric and monomeric forms of β-globin, as well as α-globin are shown in Table I. The relative ratios standardized to Hb A of the initial rates of change for α:β:β-β:Hb A were 33.5:7.4:6.4:1, respectively. These results indicate that β-β dimerization has little effect on promoting thermal stabilization over that of unmodified β-globins; however, assembly of Hb A tetramers increases stability over that of individual monomeric α- and β-globins. We have established a method to obtain pure, recombinant, soluble, authentic human β-globin chain using an E. coli. expression system. Usually, β-globin chains are isolated in low yield from human tetrameric Hb A and require dissociation of soluble stable tetramers to unstable monomers (11Ikeda-Saito M. Inubushi T. Yonetani T. Methods Enzymol. 1981; 76: 113-121Crossref PubMed Scopus (20) Google Scholar). This thermodynamically disadvantageous step is accompanied by high amounts of precipitation during purification, resulting in a very low yield of single chains. Hoffman et al. (4Hoffman S.J. Looker D.L. Roehrich J.M. Cozart P.E. Durfee S.L. Tedesco J.L. Stetler G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8521-8525Crossref PubMed Scopus (220) Google Scholar) recently developed a bacterial expression system in which human α- and β-globin cDNAs are coexpressed in the same plasmid vector in E. coli. The α- and β-globin chains are folded properly in vivo and assembled into soluble tetrameric Hb A molecules in E. coli. However, expressed chains retained N-terminal methionines. They also attempted to express β-globin cDNA alone under the same conditions and found that the expressed protein was primarily insoluble and that proper folding and heme insertion did not occur. From these results they suggested that heme was not incorporated into β-globin and that interaction between α- and β-globin chains may affect correct folding of the two globin polypeptides in E. coli (4Hoffman S.J. Looker D.L. Roehrich J.M. Cozart P.E. Durfee S.L. Tedesco J.L. Stetler G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8521-8525Crossref PubMed Scopus (220) Google Scholar). This result is similar to attempts at reconstitution in vitro to form tetrameric Hb A, as described above. It is also noteworthy that our previous attempts were unsuccessful at expressing β-globin chains alone using a yeast expression system (3Adachi K. Konitzer P. Lai C.H. Kim J. Surrey S. Protein Eng. 1992; 5: 807-810Crossref PubMed Scopus (33) Google Scholar) in which α-globin cDNA was deleted from the expression vector. This yeast system, which was originally developed by Wagenbach et al. (2Wagenbach M. O'Rourke K. Vitez L. Wieczorek A. Hoffman S. Durfee S. Tedesco J. Stetler G. Bio/Technology. 1991; 9: 57-61Crossref PubMed Scopus (111) Google Scholar), produces authentic soluble α- and β-globin chains that assemble in yeast to form soluble tetrameric human hemoglobin without N-terminal methionine (2Wagenbach M. O'Rourke K. Vitez L. Wieczorek A. Hoffman S. Durfee S. Tedesco J. Stetler G. Bio/Technology. 1991; 9: 57-61Crossref PubMed Scopus (111) Google Scholar, 3Adachi K. Konitzer P. Lai C.H. Kim J. Surrey S. Protein Eng. 1992; 5: 807-810Crossref PubMed Scopus (33) Google Scholar). The method we now describe to express soluble authentic human β-globin should facilitate production and isolation of single-chain variants in order to further study mechanisms of folding of single chains and assembly. The original pHE2 plasmid was engineered to coexpress α- and β-globin chains in addition to methionine aminopeptidase under control of a taq promoter (6Shen T-J. Ho N.T. Simplaceanu V. Zou M. Green B.N. Tam M.F. Ho C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8108-8112Crossref PubMed Scopus (138) Google Scholar, 10Ben-Bassat A. Bauer K. Chang S.-Y. Myambo K. Boosman A. Chang S. J. Bacteriol. 1987; 169: 751-757Crossref PubMed Google Scholar). The current expression system uses pHE2β to produce only β-globin and was engineered by deleting α-globin cDNA from the original pHE2 plasmid. This vector system produces significant amounts of soluble β-globin chains that lack N-terminal methionine and contain monomeric and dimeric forms of β-globin. Conditions for expressing β-globin chains alone are the same as those reported previously (6Shen T-J. Ho N.T. Simplaceanu V. Zou M. Green B.N. Tam M.F. Ho C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8108-8112Crossref PubMed Scopus (138) Google Scholar). Expressed β-globin chains bound heme and spectrophotometric properties were similar to that of authentic β-globin from human red blood cells. These results suggest that conditions in bacteria promote correct folding to produce soluble β-globin chains in the absence of α-globin chain expression. Dimeric as well as monomeric forms of β-globin were obtained using this E. coli expression vector system. Dimers formed from monomers via an intermolecular disulfide bond between cysteine residues, and this bond could be reversed by addition of DTT, even though monomer yield in each experiment was variable. The finding of oxidized dimeric forms implies that cysteine and methionine residues underwent oxidation either in bacteria or during purification. Spontaneous conversion of monomeric β-globin to the disulfide bonded dimer is also readily apparent for native β-globin chains from human red blood cells. Furthermore, some of the purified β-globin chains prepared from human red blood cells upon storage or in the presence of oxidants also showed formation of a doublet dimer band of approximately 32 kDa like recombinant β-globin chains. Human β-globin chain has two cysteine residues (β93 and β112) which could theoretically participate in intermolecular disulfide bond formation. Our tryptic peptide results suggest that Cysβ112 is involved in disulfide bond formation between two β-globin chains, even though other cysteine interactions can not be excluded at this point. It is known that isolated β-globin chains aggregate to form β4 homotetramers, while isolated α-globin chains only form dimers (23Valdes Jr., R. Ackers G.K. J. Biol. Chem. 1977; 252: 74-81Abstract Full Text PDF PubMed Google Scholar). Recent x-ray analysis of β4 hemoglobin at 1.8-Å resolution indicated that Cysβ112(G14) is located at the β-chain interface and that the side chains of Cysβ1112 and Cysβ2112 in the β4 tetramer are very close to the molecular dyad at the β1β2 interface (24Borgstahl G.E.O. Rogers P.H. Arnone A. J. Mol. Biol. 1994; 236: 817-830Crossref PubMed Scopus (52) Google Scholar). These two residues exist on the surface of the β4 tetramer and may be involved in weak interactions with other residues. In the presence of oxidants, either in vivo or in vitro, these two cysteines could readily form a disulfide bond. Furthermore, it is known that Cysβ112 is located at the interface of α1β1 in hemoglobin tetramers and that this residue also interacts with Valα107 (G14) (24Borgstahl G.E.O. Rogers P.H. Arnone A. J. Mol. Biol. 1994; 236: 817-830Crossref PubMed Scopus (52) Google Scholar). These structural observations are consistent with our results and conclusions. Our results show that monomeric β-globin expressed in bacteria assembled in vitro with α-globins as readily as native human β-globin, while dimeric β-globin chains do not form tetramers upon addition in vitro of α chains. The disulfide bond formed between two β-globin chains does not allow dissociation into monomers without reducing agents such as DTT. These results also suggest that β-β dimer formation can be prevented by α-globin chains and that Cysβ112 then can participate in a hydrophobic interaction with Valα107(G14), which facilitates αβ dimer formation and generates stable tetrameric hemoglobin. It is well known that monomeric α- and β-globin chains are not as stable as Hb A. Isolated α- and β-globin chains are also more readily denatured upon mechanical agitation than Hb A tetramers (19Asakura T. Adachi K. Sono M. Friedman S. Schwartz E. Biochem. Biophys. Res. Commun. 1974; 57: 780-786Crossref PubMed Scopus (20) Google Scholar). Calculations of electrostatic free energy contributions showed that tetramer formation was more preferable than the monomeric forms (20Friend S.H. Matthew J.B. Gurd F.R.N. Biochemistry. 1981; 20: 580-586Crossref PubMed Scopus (23) Google Scholar). Our present thermal stability experiments demonstrate that both α- and β-globin chains are significantly denatured after being exposed to heat at 60°C and that α-globins are less stable than β-globins. The thermal unfolding kinetics of Hb A and its subunits measured by temperature-induced changes in ellipticity at 222 nm in the CD spectra under the present experimental conditions at neutral pH follow first-order kinetics. Acid and alkali denaturation studies of α and β subunits as well as Hb A have been reported previously and show that the denaturation process follows complex first-order kinetics (25Peruts M.F. Nature. 1974; 247: 341-344Crossref PubMed Scopus (41) Google Scholar, 26Franchi D. Fronticelli C. Bucci E. Biochemistry. 1982; 21: 6181-6187Crossref PubMed Scopus (14) Google Scholar). The change for the disulfide bonded β-β dimer was slightly less than that of monomeric β-globin chains, indicating that dimers are slightly more stable than monomers. Formation of tetrameric Hb A was, however, most stable to temperature-induced changes in CD spectrum. Monomeric βSH-globin chains can form β4 tetramers that are more stable forms than the monomeric form (23Valdes Jr., R. Ackers G.K. J. Biol. Chem. 1977; 252: 74-81Abstract Full Text PDF PubMed Google Scholar, 27Philo J.S. Lary J.W. Schuster T.M. J. Biol. Chem. 1988; 263: 682-689Abstract Full Text PDF PubMed Google Scholar). However, β4 is unstable compared with tetrameric Hb A, even though the quaternary structure of β4 tetramers resembles that of the α2β2 tetramer (24Borgstahl G.E.O. Rogers P.H. Arnone A. J. Mol. Biol. 1994; 236: 817-830Crossref PubMed Scopus (52) Google Scholar). These interactions are entropy-droven and involve predominantly hydrophobic interactions. The β4 tetramers readily dissociate into monomeric forms upon addition in vitro of α-globins (28Bunn H.F. McDonald M.J. Nature. 1983; 306: 498-500Crossref PubMed Scopus (64) Google Scholar). In contrast, α chain alone can form dimers, which are much more unstable than β-globins. Assembled α2β2 tetramer, however, promotes stability, while β-β dimer, which cannot assemble with α-globin, readily precipitates like β-globin. It would be interesting to know whether dimeric forms exist in red blood cells from patients with various forms of α thalassemia, which result in unbalanced production of α-globin chains (29Schwartz E. Benz Jr., E.J. Hoffman R. Benz Jr., E.J. Shattil S.J. Furie B. Cohen H.J. Hematology. Churchill Livingstone Inc., New York1991: 368Google Scholar). We thank Drs. Margaret Keller, Eric Rappaport, and members of the Nucleic Acid/Protein Core at the Children's Hospital of Philadelphia for automated DNA sequence analysis. We are grateful to Drs. C. Ho and T-J. Shen (Carnegie Mellon University, Pittsburgh, PA) for providing the pHE2 plasmid. We also acknowledge John Kim and Cedric H. L. Shackleton (Children's Hospital Oakland Research Institute Mass Spectrometry Facility) for skillful technical assistance and discussion as well as support in mass spectrometric studies, respectively." @default.
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