Molecular aspects of embryonic hemoglobin function

Molecular Aspects of Medicine 23 (2002) 293–342 www.elsevier.com/locate/mam

Review

Molecular aspects of embryonic hemoglobin function Thomas Brittain

*

School of Biological Sciences, The University of Auckland, Auckland, New Zealand

Abstract In order to provide the appropriate level of oxygen transport to respiring tissues, we need to produce a molecular oxygen transporting system to supplement oxygen diffusion and solubility. This supplementation is provided by hemoglobin. The role of hemoglobin in providing oxygen transport from lung to tissues in the adult is well-documented and functional characteristics of the fetal hemoglobin, which provide placental oxygen exchange, are also well understood. However the characteristics of the three embryonic hemoglobins, which provide oxygen transport during the first three months of gestation, are not well recognized. This review seeks to describe the state of our understanding of the temporal control of the expression of these proteins and the oxygen binding characteristics of the individual protein molecules. The modulation of the oxygen binding properties of these proteins, by the various allosteric effectors, is described and the structural origins of these characteristics are probed. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Hemoglobin; Embryonic; Development; Function; Structure Abbreviations: CO, Carbon monoxide; 2,3 D.P.G., 2,3 Diphosphoglycerate; eIF2, Eukaryotic initiation factor; EKLF, Erythroid Krupple like factor; Hb, Hemoglobin; SSP, Stage selector protein

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 2. General features of globin genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 2.1. Chromosomal organization and structure of the globin genes . . . . . . . . . . . 295 2.2. Major control elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 3. Globin gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

*

Fax: +64-9-3737414. E-mail address: [email protected] (T. Brittain).

0098-2997/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 9 8 - 2 9 9 7 ( 0 2 ) 0 0 0 0 4 - 3

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3.1. 3.2. 3.3. 3.4. 3.5.

Function of LCR and HS-40 Transacting factors . . . . . . . . mRNA stability . . . . . . . . . . The role of heme . . . . . . . . . Hemoglobin assembly . . . . . .

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4. Developmental hematopoiesis and globin 4.1. Developmental hematopoiesis . . . . . 4.2. Gene switching . . . . . . . . . . . . . . . 4.3. Reactivation of fetal genes . . . . . . .

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298 299 300 300 301

gene switching . . . . . . . . . . . . . . . . . . . . ............................... ............................... ...............................

302 302 303 305

5. Hemoglobin structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 5.1. Globin evolution and primary structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 5.2. Secondary, tertiary and quaternary structure . . . . . . . . . . . . . . . . . . . . . . . . . 307 6. Hemoglobin function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Homotropic effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Quantitative description of hemoglobin function . . . . . . . . . . . . . . . . . . . . . . 6.3. Heterotropic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

310 310 311 312

7. Embryonic hemoglobin function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Homotropic effects in human embryonic hemoglobins. . . . . . . . . . . . . . . . . . . 7.2. Heterotropic effects in human embryonic hemoglobins . . . . . . . . . . . . . . . . . . 7.3. CO toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Redox activity in embryonic hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. Heme binding and dimerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. Kinetic studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1. Ligand binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.2. Subunit dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

314 315 317 321 322 324 324 324 326

8. Embryonic hemoglobin structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Embryonic hemoglobin Gower II structure . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. The structure of c globin subunits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. f globin structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

326 327 328 329

9. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

1. Introduction As a consequence of its high energy yield, aerobic oxidation, directly or indirectly, drives almost all metabolic processes in humans. Oxygen solubility in body fluids is insufficient to provide the necessary oxygen flux for the maintenance of aerobic metabolism and as a consequence an efficient oxygen transport system is required. Such an oxygen transport system necessarily requires three components, namely,

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an efficient gas transfer surface, an oxygen binding molecule to increase oxygen solubility and a pump to move bulk solution around the body. During human development, with the exception of the first few weeks of gestation, the heart provides the necessary pump activity. The gas exchange surface alters significantly during development changing from body surface, to placenta, to lungs, as development progresses. On the other hand, additional oxygen solubility is provided at all stages of development by the presence of the molecular oxygen transporting protein hemoglobin. As this molecule requires properties optimized for oxygen transport under the vastly different physiological conditions encountered during the embryonic, fetal and adult phases of human development it is necessary for the body to synthesize different forms of the protein, with characteristics appropriate to the situation in which the developing individual finds itself. Hence the individual, as an adult, produces hemoglobin optimized for pulmonary/tissue oxygen transport, a fetal protein for placental/tissue oxygen transport and a series of three embryonic proteins to cope with the extremely rapidly changing environment presented by the first 12 weeks of human development. In particular, these three embryonic proteins need properties which allow them to provide efficient oxygen transport ranging from diffusional oxygen scavenging from the amniotic fluid through to the transition to placental circulation. Although the embryonic phase of development lasts for only approximately 14 weeks, this period encompasses the most dramatic changes in physiology and physiognomy the individual must face during the whole of its life. Thus three different hemoglobins are required to provide the range of oxygen binding characteristics necessary to span this period of ever-changing demands. Surprisingly Nature satisfies all of these requirements, at all stages of human development, by employing a single protein architecture capable of providing the necessary range of oxygen binding characteristics by just a few amino acid substitutions within the hemoglobin molecule. We are only now beginning to understand the structural origins of the modulation of the oxygen binding characteristics of the three human embryonic hemoglobins.

2. General features of globin genes 2.1. Chromosomal organization and structure of the globin genes The globin proteins, which constitute the subunits of hemoglobin are encoded in genes which exist in two clusters on chromosomes #11 and #16 (Fig. 1) (Diesseroth et al., 1977, 1978). The a cluster, which includes the f; wf; wa and a2 and a1 genes, covers approximately 45 kbp of DNA and lies in a GC rich area in the telomeric region of chromosome #16. This region is associated with unmethylated CpG islands, is constitutively early replicating and is accessible in many cell types (Flint et al., 1997; Vyas et al., 1992; Smith and Higgs, 1999; Brown et al., 2001; Hardison et al., 1997). The b globin cluster, which includes the e; cG ; cA ; wb; d and b genes, covers approximately 70 kbp of DNA and lies within an AT rich region on chromosome #11.

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Fig. 1. Chromosomal organization of the globin genes and upstream control elements. Open triangles represent expressed genes. Black triangles represent non-expressed pseudo-genes. Grey triangles represent genes expressed at only low levels. Numbers below lines represent base pairs from the first base 50 to the first globin gene in each complex.

This region of DNA shows characteristics typical of tissue specific expression (Hardison, 1998; Engel and Tanimoto, 2000). The genes in each cluster are organized in a 50 –30 sequence, which matches their expression sequence during human development. The globin genes themselves share a number of common characteristics. The genes are typically 1–2 kbp long and are composed of three exonic and two intronic segments. In the a genes exon 1 codes for the first 31 amino acids of the globin protein. This is followed by an intron of somewhat variable length ð117 bp in a, 1265 bp in fÞ. Exon 2 codes for amino acids 32–99. This is followed by intron 2 which has a more defined length of 140–149 bp in a and 341 in f. The final exon codes for amino acids 100–141 of the a globin proteins. In the b cluster the structure of the globin genes is less variable than in the a cluster with the first exon coding for amino acids 1–30. This is followed by the first intron which is 122–130 bp in length and is followed by exon 2 which codes for amino acids 31–104. The second intron is 850– 904 bp in length and is followed by exon 3 which codes for amino acids 105–146 of the b globin proteins (Nienhuis and Maniatis, 1987). There is strong evidence that the three exonic segments code for amino acid sequences which serve distinct functions in the mature protein. The product of exon 1 contains the amino acids largely responsible for the interaction of hemoglobin with protons and organic phosphates. Exon 2 codes for the peptide which binds the heme group and which contains the amino acids primarily responsible for the creation of the a1 b2 intersubunit interface contact and hence dimer co-operativity. The third exon codes for a peptide which contains the amino acids involved in the a1 b1 contact surface and hence co-operativity within the fully assembled tetrameric protein (Eaton, 1980; Blake, 1981; Go, 1981; Craiks et al., 1981). Each gene is preceded by a 50 promoter sequence and followed by a 30 polyadenylation sequence. The promoter sequence consists of an AT rich region approximately 30 bp upstream of the CAP site and contains a TATA box (Efstratiadis et al., 1980). A second promoter region 70–90 bp 50 to the CAP site contains the CCAAT

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box (Mellon et al., 1981; Filipe et al., 1999). A CACCC sequence is present 90–105 bp upstream of the CAP site in the b globin genes (Jane and Cunningham, 1998) whilst the a genes contain a CCGCCC repeat (Nienhuis and Maniatis, 1987). 2.2. Major control elements In addition to the usual promoter sequences alluded to above, the globin gene clusters also possess other major upstream control elements which interact with the genes within the cluster to provide controlled, sequential expression. The b gene cluster, which is the best characterized, possesses a cis-regulatory element 30–50 kbp upstream from the cluster known as the Locus Control Region (LCR). (Fig. 2) (Hardison et al., 1997; Tuan et al., 1989; Forrester et al., 1986, 1987). This structure consists of an insulator sequence (HS5) and four DNAse I nuclease hypersensitive structures (HS1-4) which are erythroid specific (Grosveld et al., 1987). The analogous structure within the a gene cluster is known as HS-40 and consists of a group of elements 40 kbp upstream of the a cluster (Fig. 2) (Higgs et al., 1990; Jarman et al., 1991). The LCR confers tissue-specific, copy number dependent high level b globin protein synthesis (Townes and Behringer, 1990; Grosveld et al., 1993a). The HS-40 on the other hand does not ensure copy number dependent expression of the a globin genes (Sharpe et al., 1992; Sharpe et al., 1993; Robertson et al., 1995). These differences in function have been considered to reflect the different chromatin context in which the globin gene clusters are found (Craddock et al., 1995; Orkin, 1995). Within both the LCR and HS-40 control regions the active regions consist of 200– 300 bp sequences encompassing a DNAse I hypersensitive site and these core regions contain motifs typical of nuclear regulatory proteins binding sites (Strauss et al., 1992; Courney and Tijian, 1998; Martin et al., 1990; Andrews et al., 1993; Lee et al., 1987; Chan et al., 1993; Moi et al., 1994; Ho, 1999). The exact positioning of the various binding motifs has been mapped to the individual DNase I hypersensitive sites. The b globin gene cluster also possesses a downstream enhancer approximately 600–900 bp 30 of the polyadenylation signal (Trudel and Contantini, 1987; Behringer et al., 1987) and an enhancer sequence embedded in intron 2. Negative, silencing elements have been identified in the b gene and two upstream silencers have been reported in the c gene (Gumucio et al., 1994).

Fig. 2. Binding motifs within each control element. Black squares represent GATA boxes whilst black ovals represent AP1-NF-E2 motifs.

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3. Globin gene expression 3.1. Function of LCR and HS-40 Globin gene expression occurs sequentially in yolk sac derived cells during the embryonic phase of development, liver and spleen cells during the fetal stage and bone marrow cells during the adult stage of human development. Whatever the site of globin synthesis there is a requirement for a high level of specific protein synthesis within a specific cell type. The process of gene expression is controlled at various levels, namely, chromatin structure, transcription and translation. It is well recognized that globin protein synthesis occurs within the context of open, non-methylated euchromatin structures which are provided primarily by the interaction of the specific globin genes with either the LCR or the HS-40 structures. Most studies are consistent with the idea that the HS regions of the LCR function in unison to produce a holocomplex which is necessary for full expression of the LCR activity (Ho, 1999; Grosveld et al., 1989). However, particular functions have been associated with specific HS regions. HS1, which is located 6 kbp 50 to the first gene of the b globin cluster ðeÞ confers position – independent expression in transgenic mice, but does not increase expression levels of b globin (Fraser et al., 1990, 1993). In fact, HS1 function may be dispensable, as deletion of this region from the LCR does not produce any significant hematological defects (Kuzolik et al., 1991). HS2 confers position independent high level globin expression in transgenic mice where it exhibits classical enhancer activity (Ryan et al., 1989; Talbot et al., 1989; Morley et al., 1992; Tuan et al., 1989). Within the naturally occurring LCR it appears that other portions of the LCR can substitute for HS2 activity on deletion (Hardison et al., 1997). HS3 is the most active region in producing high level expression of b type globin proteins and is particularly active in providing high level expression of the c globin protein during the embryonic and fetal stages of development (Fraser et al., 1990, 1993). Deletion of HS3 in transgenic mice causes a reduction in expression of all linked globin genes (Bungert et al., 1995; Milot et al., 1996; Petersen et al., 1996). Within the LCR the main chromatin opening activity has been mapped to the HS3 region (Hardison et al., 1997; Ellis et al., 1996), although full chromatin opening activity appears to require the entire LCR and a number of other remodeling activities such as SWI/SNF and histone acetyl transferases within the erythroid cell (Cote et al., 1994; Peterson and Tamkun, 1995; Brownell et al., 1996; Kadonaga, 1998; Armstrong et al., 1998; Lopez et al., 2002). HS4 by contrast has its greatest impact on b globin synthesis during the adult stage in transgenic mice, but little effect at the embryonic or fetal stages of development. HS5 appears to perform the role of insulator for the LCR (Li and Stamatoyannopoulos, 1994; Yu et al., 1994). There is mounting evidence that the achievement of synergy between the various HS regions is mediated by the intervening sequences of DNA and full activity of b type globin gene expression is only achieved in the presence of all the HS elements (Jackson et al., 1996a,b). The a globin gene cluster has been shown to contain DNase I hypersensitive sites 4, 8, 10, 33 and 40 kb upstream from the f CAP site (Higgs et al., 1990; Jarman et al.,

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1991). Of these sites only the sequence of DNA 40 kb upstream of the CAP site appears to have significant effects on a globin gene expression (Higgs et al., 1990). As might be expected, in that it only provides a single gene switching function (f–a) and carries out its functions within a permanently open chromatin environment, the analogous HS-40 control element does not appear to function in exactly the same way as the LCR (Higgs et al., 1990; Huang et al., 1998). Nevertheless, the HS-40 contains six nuclear factor binding motifs (four GATA-1 and two AP-1 motifs; Jarman et al., 1991) contained within five DNase hypersensitive sites analogous to those in the LCR (Higgs et al., 1990). The HS-40 exhibits both positive and negative regulatory effects on the f-globin promoter (Huang et al., 1998).

3.2. Transacting factors The interaction of promoters and control regions with the specific globin genes is primarily mediated by an array of DNA binding proteins. Within the LCR cores and HS sites three major protein binding motifs are evident, namely those for GATA, AP-1 and CACC binding proteins (Orkin, 1990; Huang et al., 1998). The position independent activity of the LCR correlates best with the presence of GATA and CACC motifs (Ellis et al., 1993; Philipsen et al., 1993), whilst the classical enhancer activity, particularly of HS2 requires the APO-1 motif for expression. The various transcription factors have been studied in detail. It has become clear that not only do these factors interact individually with various control sequences within the genome, but also interact with each other and themselves to yield a very complex pattern of gene regulation. A myriad of protein factors are clearly responsible for gene expression and developmental switching. Of these the best understood are reviewed below. GATA sequences are found throughout the cores of the LCR and HS-40 as well as in proximal regulatory regions of the f; e and c, but not a; d or b genes (Orkin, 1990; Ikonomni et al., 2000). The proteins responsible for binding to these GATA regions are referred to as the GATA proteins of which GATA-1 and GATA-2 appear to be the most significant. The GATA proteins are of approximately 50 kDa mass and contain zinc finger binding domains in their C termini which bind to the minor groove of the DNA (Tsai et al., 1989; Orkin, 1995). GATA-1 is a potent transcriptional activator and is multiply phosphorylated on Ser residues (Crossley and Orkin, 1994). GATA-1 is first expressed in all multipotent stem cells but is only maintained at high levels in committed erythroid cells. GATA-2 is structurally very similar to GATA-1 (Cairns et al., 1994; Sposi et al., 1992). Specific globin gene expression appears to require GATA-1 self interaction or interaction with other factors such as Sp1 and EKLF. EKLF is a 48 kDa, three zinc finger, Kruppel-domain protein which recognizes the CACCC motif to which it binds with high affinity (Miller and Bieker, 1993). In this way it binds directly to HS3 of the LCR and promotes b globin synthesis much more than c globin synthesis (Terwari et al., 1998; Donze et al., 1995; Perkins et al., 1996; Guy et al., 1998). EKLF expression is controlled by a GATA sequence

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containing promoter and so is itself controlled by the concentration of GATA-1 (Crossley et al., 1994). EKLF is equally expressed in both embryonic and definitive hematopoietic cells although it is not absolutely required for embryonic erythropoiesis (Perkins et al., 1995; Orkin, 1995). Also important, but less well characterized, are NF-E2 and stage selector protein (SSP). NF-E2 is a hetero dimeric protein (Andrews et al., 1993; Igarashi et al., 1994) which binds strongly to the AP-1 like motif of HS2, but also elicits activity at HS3 and HS4 as well as HS-40 (Talbot et al., 1990; Mignotte et al., 1989; Ney et al., 1990). NF-E2 is required for maximal globin gene expression but its activity may be replaced by other protein factors. SSP, through its binding to the stage selector element (SSE) in the c promoter, has an activity which is specific to the stage of development (Jane et al., 1992). It is enriched in fetal and embryonic erythrocytes. 3.3. mRNA stability The high concentration level of hemoglobin which is achieved within erythrocytes is, in part, made possible by the unusual stability of globin mRNA. b mRNA is highly stable, exhibiting a cellular half life in excess of 60 h (Lowenhaupt and Linger, 1978; Volloch and Housman, 1981). Much of its stability can be traced to the polyadenylation tail of the mRNA, which probably acts as a buffer towards the action of exonucleases involved in normal mRNA degradation (Sachs, 1993; Ross, 1995; Bernstein et al., 1989; Ford et al., 1997). Added stability in both a and b mRNA appears to reside in the 30 UTR and 50 UTR (Wang et al., 1995; Weiss and Liebhaber, 1994). 3.4. The role of heme In order to obtain fully functional hemoglobin molecules not only is there a requirement for matched synthesis of the constituent a and b type globin apo-proteins, but also the co-ordinated synthesis of the heme prosthetic group. The co-ordinated synthesis of heme is particularly important as heme is very toxic. Indeed, the concentration of heme within the cell is very tightly regulated by multiple feed back mechanisms in which free heme drastically curtails its own synthesis. Heme plays a major role also in the co-ordination of the synthesis of hemoglobin at the level of protein translation. Protein synthesis normally occurs after disassembly of the 40S and 60S ribosomal subunit complex. This assembly is controlled by the action of the initiation complex. The pre-initiation complex is produced by the interaction of GTP, Met-tRNA and the eIF2 protein complex with the 40S ribosomal subunit (Jagus et al., 1981; Moldave, 1985). The eIF2 complex binding activity is prevented by phosphorylation of the protein on Ser 51 of the a subunit (DeBenedetti and Baglioni, 1983). Phosphorylation of the eIF2 protein by ATP occurs via the action of the HCI kinase (heme controlled inhibitor) or HRI (heme regulated inhibitor). Heme binds to the kinase inactivating it, probably by promoting inter-subunit disulphide

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Fig. 3. Heme control of globin expression. A cartoon showing the effect of heme on globin expression exerted at the level of the HCI phosphorylation of eIF2.

bond formation between HRI molecules (Chen and London, 1995; Safer et al., 1982; Safer, 1983). Thus the overall action of this multi-tiered control mechanism is such that an excess of heme activates globin protein synthesis. In this complex manner the cell ensures a balanced synthesis of apo-globin proteins and heme (Fig. 3). 3.5. Hemoglobin assembly The assembly of globin subunits from their constituent apo-globin protein and heme has been found to occur rapidly and spontaneously in vitro (Gibson and Antonini, 1963; Rose and Olson, 1983; Hargrove and Olson, 1996). Quaternary assembly of the heme containing globin subunits also occurs rapidly and spontaneously in vitro (Antonini et al., 1966; Antonini and Brunori, 1971). However, detailed analysis of the in vitro reaction of heme with apo-globin proteins shows that the initial product exists as a 50:50 mixture of molecules, in which the heme group takes up one or other of the two possible rotameric configurations in which there is 180° rotation about the porphyrin a–c meso axis (LaMar et al., 1985; Yamamoto and LaMar, 1986; Jue and LaMar, 1984). It should be noted however, that there is no experimental evidence to suggest that these rotamers have any different oxygen binding properties. The rotamers equilibrate to yield predominantly a single form, over a period of up to weeks, depending on the heme iron oxidation and ligation state. The final product formed in vitro is indistinguishable from that contained in circulating mature erythrocytes. It was long assumed that a similar process of formation and subsequent equilibration occurred during the natural synthesis and assembly of hemoglobin in vivo. However, recent NMR studies on the newly synthesized hemoglobin obtained from purified populations of reticulocytes indicates that heme insertion into apo-globin in erythrocyte precursors, at least in part, occurs into nascent apo-globin proteins, as they are synthesized on the ribosome (Mathews and Brittain, 2001). Support for this proposal is found in a previous study employing radio-labeled heme insertion (Komar et al., 1993).

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4. Developmental hematopoiesis and globin gene switching 4.1. Developmental hematopoiesis During the first two weeks of human life the individual is small enough to meet its oxygen supply demands by means of simple diffusion processes, obtaining oxygen from the maternal interstitial fluid. From day 15 of gestation simple diffusion is no longer sufficient to meet the metabolic demands of the growing embryo and the first globin genes are activated to produce embryonic hemoglobin, which acts as an oxygen carrier (Hemoglobin Gower I f2 e2 , Fantoni et al., 1981). This process begins as the mesenchymal cells of the yolk sac begin to differentiate to produce embryonic red blood cells. These cells, which are large ðvol approximately 200 l3 Þ (Stamatoyannopoulos and Nienhuis, 1987) nucleated spherical cells, initially form blood islands within the yolk sac prior to their migration down the vitelline vessels to the developing embryo. At this stage the venous and arterial circulations are not joined and blood movement is pulsatile, driven by a tubular heart. By the middle of week four of gestation the heart becomes septated and a true circulation is established. During this phase two other embryonic hemoglobins are synthesized ðHb Gower II a2 e2 , Hb Portland f2 c2 Þ and the placenta begins to develop. Thus up to day 37 of gestation yolk sac derived red blood cells are found to contain predominately Hb Gower I (f2 e2 , Hetch et al., 1966). The relative proportion of Hb Gower I, Gower II and Portland at this point are 42%, 24% and 21%, respectively. Successively as the f to a switch occurs, Hb Gower II ða2 e2 Þ begins to dominate. Over the next six weeks there is a gradual shift in the significance of the two circulatory systems with the predominantly vitelline circulation giving way to the placental circulation as the yolk sac disappears. It is during this phase that the liver, and to a lesser extent the spleen, takes over as the major sites of hematopoiesis producing definitive discoid, annucleate red blood cell ðapproximate vol 125 l3 Þ containing fetal hemoglobin ðHbF, a2 c2 Þ and traces of adult hemoglobin (Bloom and Bartelmez, 1940; Pataryas and Stamatoyannopoulos, 1972; Wintrobe, 1981). Later still, from approximately twenty weeks gestation, the bone marrow begins to produce definitive discoid, annucleate red blood cells ðapproximate vol 80 l3 Þ containing adult hemoglobin (HbA, a2 b2 , Lewis, 1974). Together with the change in hemoglobin contained within the red blood cells, the change in red blood cell size and shape facilitates rapid oxygenation during passage through the placenta and lungs (Brittain and Simpson, 1989). The fetal and adult red blood cells also differ in some important biochemical aspects. The fetal red cell exhibits the i surface antigen whilst the adult red blood cell exhibits the I antigen. The fetal erythrocyte also shows lower levels of glycolytic enzymes and different isozyme profiles compared to the adult red blood cell (Stamatoyannopoulos and Nienhuis, 1987). At birth the neonate has a circulation consisting of red blood cells containing approximately 70% fetal and 30% adult hemoglobin. Within five months the fetal hemoglobin content of the infants blood falls to around 3% and after two years is completely replaced with bone marrow derived adult hemoglobin, with a minor component of HbA2 (3%, a2 d2 , Huehns, 1974). Thus, during human development in utero we see a progression of globin synthesis, which produces a

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Fig. 4. Hematological development. The age dependence of the site of hemoglobin synthesis (a), globin chain expression (b), hemoglobin synthesis (c) and red blood cell type (d) is shown. Numbers represent weeks of gestation.

series of hemoglobins optimized for oxygen transport under the ever changing physiological conditions posed by the developing fetus and its inter-uterine environment (Fig. 4). 4.2. Gene switching The synthesis of the series of hemoglobins is made possible by the process of gene switching (Jane and Cunningham, 1996; Grosveld et al., 1993a; Orkin, 1995;

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Stamatoyannopoulos and Nienhuis, 1987). The control of gene expression within the globin gene clusters occurs primarily at the level of transcription, but does not appear to have a universal mechanism. There is good evidence that the expression of the earliest globin genes, namely f and e are autonomously regulated and the switching off of these genes does not require competition with any other globin genes (Pondel et al., 1992; Shih et al., 1990; Dillon and Grosveld, 1991). Gene activity appears to require the interaction of the LCR or HS40 with promoter sequences of the earliest genes and further involves interactions with some of the transacting factors described above. Initial activation of the embryonic f globin gene requires the interaction of GATA-1 and NF-E2/AP1 with the HS40 control region (Zhang et al., 1993; Sabath et al., 1995). As development progresses the switch from e to c appears to involve the synthesis and binding of silencer elements to the e gene promoter, which frees the LCR for interaction with the c gene promoter (Li et al., 1998; Raich et al., 1992, 1995; Peters et al., 1993; Perez-Stables, 1994; Li et al., 1989). The synthesis of the e globin is associated with high levels of GATA-2. However, increasing levels of GATA-1 suppresses the expression of GATA2 and leads to a decrease in the level of e globin mRNA, but has no effect on the levels of expression of c; f, or a (Ikonomni et al., 2000). Much of this interaction seems to be favored simply by the physical proximity of the c; as opposed to the b gene, to the LCR (Handscombe et al., 1991; Peterson and Stamatoyannopoulos, 1993; Grosveld et al., 1993a; Grosveld et al., 1993b). The next switch from c to b globin gene transcription is clearly a competitive event, controlled not only by the distance of the gene from the LCR, but also by the changing concentrations of the various transacting proteins (Orkin, 1995). The changing concentrations of these factors appear not to produce an allor-none binding to the c or b promoter, but rather the shift in concentration of the factors appears to lead to a changing frequency of interaction with the two competing promoters (Wiejgerde et al., 1995). Obviously, the developmental pattern of a type globin gene expression requires only a single gene switching event. It appears that in this case the interaction of HS-40 with the f globin gene is interrupted during the f to a switch, when the changing concentrations of NF-E2 and the GATA proteins lead to a change in promoter affinity which favors the a genes (Huang et al., 1998; Zhang et al., 1995) (Fig. 5). The foregoing discussion of gene switching is based on the shift from one gene type to another. However, there are multi-copies of many of the globin genes and this leads to an additional subtlety in terms of globin expression. During a switching event the dual copies of any particular globin type are not necessarily expressed at the same level. The c globin protein is encoded by two copies of the c gene (G c and A c). During normal fetal development the expression of the globin genes is such that the G c=A c ratio is 7:3 (Huisman et al., 1974; Huisman et al., 1977). During the adult phase approximately 0.5% of the circulating red blood cells are F cells which contain HbFða2 c2 Þ, due to incomplete repression of the c genes. In contrast to the HbF produced within the fetal phase, the HbF produced during the adult phase shows a G c=A c ratio of 2:3 (Schroeder et al., 1971; Schroeder, 1980; Boyer et al., 1975; Jensen et al., 1982; Nienhuis and Benz, 1977). By analogy the expression of the a globin protein during the embryonic and fetal stages of development is produced equally

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Fig. 5. LCR control of beta globin gene expression. The interaction of the LCR with the various beta globin genes is shown for the three developmental phases. Non-expressed genes are indicated by a black square; expressed genes by a white square and partially expressed genes by a grey square. Open circles represent a gene silencer; triangles Stage specific protein binding and cross EKLF. LCR-gene interactions are indicated by a solid arrow and competitive interactions by broken lines.

from the a1 and a2 genes. However, from the 18th week of gestation onwards there is a gradual shift in this ratio until at the adult stage of development the a1 and a2 genes are expressed in a 1:2.5 ratio (Albitar et al., 1992). This subtlety almost certainly arises from the relative physical position of the two genes with regards to the LCR and HS40 elements coupled to the time-dependent shift in concentration of the various transacting proteins. 4.3. Reactivation of fetal genes Despite all of these complex control mechanisms it has been found that administration to adults of such simple organic chemicals as 5-azacytidine, hydroxyurea or butyrates can lead to re-activation of the c globin genes (DeSimone et al., 1982; Ley et al., 1983, 1982; Charache et al., 1995; Perrine et al., 1988, 1989). This finding is of fundamental importance as it holds the possibility for the treatment of a number of major b globin linked hemoglobinopathies such as b thalassaemia and sickle cell disease. In such cases reactivation of the c globin gene could potentially lead to the synthesis of fetal hemoglobin in the circulation of the adult sufferer. Although fetal hemoglobin has evolved for optimal placental/tissue oxygen transport in the fetus, at the adult stage of development, modulation of other blood parameters allows fetal hemoglobin to provide sufficient oxygen transport capacity without any major adverse effects. This situation is seen in the case of Hereditary Persistence of Fetal

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Hemoglobin (a naturally occurring condition which in some instances compensates for oxygen transport defects in b thalassaemia, Poncz et al., 1989).

5. Hemoglobin structure 5.1. Globin evolution and primary structure The family of globin proteins, which now constitutes the components of the human hemoglobins, evolved via a series of gene duplication events from an ancestral myoglobin gene following a gene duplication event approximately 800 million years ago. The present day a and b globin families split from each other approximately 450 million years ago, at the time of the separation of the cartilaginous and bony fishes (Dickerson, 1971). The a family splits further into the present day a and f branches of the globin family 300 million years ago, at the time of the origin of land vertebrates, such that the a and f globins today are the most remotely related globin proteins. Indeed, multivariate analysis highlights the relationship between the human f chain and the a chains of amphibians and fish (Van Heel, 1991). The b family splits into two branches approximately 200 million years ago, at the time of the emergence of the mammals, with one branch leading to the modern b globin protein. The other branch split approximately 100 million years ago to give the modern e and c globins (Dickerson and Geis, 1983). The outcome of this sequence of gene duplication events is that the modern day human hemoglobin proteins are composed of various combinations of pairs of globin gene products originating in the a and b globin families (Fig. 6). The a family of proteins consists of a and f globins which share 59 % amino acid sequence identity and consists of a single polypeptide chain of 141 amino acids. The b family consists of the b; d; c and e chains, which are 146 amino acids in length. The d chain is 93% identical with the b chain and is considered to be the product of a recently divergent b gene, which has corrupted transcription functions and is expressed at only 10% of the b chain and as such the d protein probably represents the product of a gene ‘‘on its way’’ to becoming a pseudo gene. The c protein is 73% identical with the b protein, whilst the e protein shows 75% identity with the b protein. The c gene exists in two copies and produces proteins which differ by one amino acid. One protein has a Gly at position 136, whilst the other has an Ala at position 136. Interestingly, since the various gene duplication events, which have led to the creation of the various globin proteins, the point mutations which have occurred are found predominantly in the first and second exons of the genes. Two of the globin proteins show post-translational modification. The f chain is Nterminally acetylated on the N-terminal Ser residue (Clegg and Gagnon, 1981; Aschauer et al., 1981). The c globin found in fetal hemoglobin is present as a mixture with approximately 15% of the globin protein is acetylated on the N-terminal Gly residue (Stegink et al., 1971). The globin proteins share five residues (Thr (a 39, b 38), Phe (a 43, b 42), Leu (a 83, b 88), His (a 87, b 92), Tyr (a 140, b 145)) present in all the globin proteins.

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Fig. 6. Evolution of globin proteins.

5.2. Secondary, tertiary and quaternary structure Although the globin proteins share variable amounts of amino acids sequence identity they all fold into essentially identical secondary structures consisting almost exclusively of a helix known as the myoglobin fold (Royer et al., 2001). The members of the a family of globins fold into seven segments of a helix (A-H; the D helix is absent), whilst the b family fold into eight segments of a helix (A-H) (Perutz et al., 1960; Baldwin, 1975). In all cases the segments of a helix are attached to each other by short segments of predominantly b turns (Fig. 7). The a helical segments of the polypeptide chains in all the globin proteins fold into compact globular tertiary structures, which are stabilized by a series of hydrophobic interactions and hydrogen bonds. Although the proteins do contain Cys residues the globin proteins do not contain disulphide bonds. The folded globin proteins possess a cleft into which the heme prosthetic group binds. The binding of the heme group further stabilizes the globin protein tertiary structure. Heme binding does not involve the formation of any classical covalent bonds; rather the heme is held in place by a large number of non-covalent interactions between the heme periphery and a number of amino acids, together with a co-ordination bond between the heme iron atom and the Ne of the proximal His residue (a 87, b 92). In this configuration the heme propionate side chains are left exposed to the surrounding solvent. Under these conditions the normally six co-ordinate ferrous iron sits in a protein pocket with a vacant ligand binding site, at which oxygen reversibly binds (the other five co-ordinating ligands being provided by the four pyrrole nitrogen atoms of the heme porphyrin ring and the proximal His N) (Fig. 8). In order for the physiologically crucial process of reversible oxygen binding to occur the heme iron atom must remain in the ferrous state and the bound oxygen molecule must be bound in a nonlinear configuration. Should this not be the case then oxygen binding rapidly leads to oxidation of the heme iron to produce the physiologically inactive met- or ferric

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Fig. 7. Structure of a hemoglobin subunit.

state. The exact mechanism whereby this oxidative process is prevented remains a point of debate. However, it seems to be generally accepted that two factors, which relate to heme pocket structure, have a significant effect on the prevention of heme iron oxidation. The bound oxygen molecule is bound to the heme iron in a bent configuration (approximately 15° from the heme normal), due primarily to the presence of a second His (distal His a 58, b 63) on the opposite face of the heme group to the iron bound proximal His, mentioned above. This residue is not bound to the iron atom but provides both steric hindrance, preventing the formation of a linear O2 –Fe structure and hydrogen bonding to the iron bound oxygen molecule, which stabilizes the structure and facilitates reversible oxygen binding to the ferrous atom. Many other amino acids which line the heme binding pocket are thought to provide an appropriate polarity to the cavity, further facilitating the reversible oxygen binding process. When oxygen binds to the heme iron atom the tertiary structure of the globin protein undergoes some structural changes. Although the protein remains essentially a globular structure on oxygen binding, the movement of certain amino acid residues is of crucial functional significance, as outlined below (Gelin et al., 1983). The hemoglobin molecule also shows quaternary structure. Each molecule consists of four globin protein subunits (2 a type and 2 b type subunits) arranged in what is essentially a tetrahedral structure with a water filled internal cavity which is continuous with the solvent. As such, the hemoglobin molecule possess a number of

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Fig. 8. Structure of the heme binding cavity. The positions of the proximal and distal heme-histidine residues are shown above and below the heme plane, respectively.

inter-subunit contacts. The most extensive interface is the a1 b1 (or a2 b2 ) contact which is formed primarily by residues a 30–36 and 103–126 and residues b 30–35 and 111–131 and involves the interaction of 34 different amino acids. These contacts involve a surface area of 1805 A2 . This interface contains many hydrophobic interactions and is essentially constant in all quaternary structures. In contrast the a1 b2 (or a2 b1 ) interface involves a surface area of 1010 A2 and consists of interactions primarily between a 38–41 and 92–96 and b 36–40 and 97–102 involving the interaction of 19 different amino acids and producing a binding energy of approximately 7.5 kcal mol1 . The aa and bb interfaces are much weaker. The a1 a2 interface involves an area of 800 A2 and involves residues 1–2 and 127–141 whilst the very weak b1 b2 interface involves a meager 350 A2 (Abraham et al., 1997). As a consequence of the differing strengths of these interfaces a quaternary structure change is associated with the oxygenation process involving the mutual rotation of ab dimers, which are otherwise structurally rigid (Perutz, 1972; Fermi and Perutz, 1981; Baldwin and Chothia, 1979; Shaanan, 1983; Fermi et al., 1984). This rotation of the dimers leads to a change in inter-subunit interactions across the a1 b2 interface. The distance between the two beta subunits is reduced. In particular, in the deoxygenated state, His b2 97 is inter-digitated between Pro a1 44 and Thr a1 41. On oxygenation His b2 97 disengages from its previous site and ‘‘ratchets’’ across the a1 b2 interface to relocate between Thr a1 41 and Thr a1 38. Interestingly Thr a 38 is invariant in all known mammalian hemoglobins (Hashimoto et al., 1993), with the exception of the embryonic f globin chain where it is substituted by a Gln (Zheng et al., 1999). Naturally

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occurring mutants at position a 38 of adult hemoglobin invariably show high oxygen affinity and lower co-operativity than the native adult protein (Hashimoto et al., 1993). It is the oxygenation linked, tertiary structure change initiated, quaternary shift between the deoxy and oxy structures of the hemoglobin molecule which provide the physiologically crucial co-operative or allosteric function of hemoglobin. Although recent structural studies have identified the existence of two oxygenated quaternary structures for adult hemoglobin (R and R2) (Smith et al., 1991; Silva et al., 1992) the functional significance of this ‘‘new’’ (R2) oxygenated form is still unclear (Janin and Wodak, 1993; Tame, 1999; Doyle et al., 1992; Schumaker et al., 1997; Srivanasan and Rose, 1994).

6. Hemoglobin function 6.1. Homotropic effects The binding of oxygen to the four heme sites within the hemoglobin molecule are not independent processes. The binding of oxygen at one site increases the affinity of the remaining sites for oxygen, leading to a positive co-operativity or allostery within the protein. This effect of bound oxygen molecules on subsequent binding of further molecules of oxygen is known as a homotropic effect. The homotropic effect manifests itself in a sigmoidal dependence of saturation vs oxygen concentration. At the phenomenological level such oxygen binding activity leads to physiological advantage, as significantly enhanced levels of oxygen off-loading can occur over a small range of oxygen concentration and can be expressed in terms of two experimentally determined parameters (p50 , h). The p50 value is the concentration of oxygen required to achieve half saturation, under some particular set of conditions, and the Hill coefficient (h) is a measure of the steepness of the oxygen binding curve at the point of half saturation and is associated with the degree of co-operative activity between the hemoglobin subunits (Hill, 1910). Using these two values alone useful comparative studies can be made on the function of hemoglobin. The homotropic effect arises from the structural changes associated with oxygen binding. This co-operative action has been interpreted in structural terms based on the three-dimensional structure of the initial deoxygenated and final oxygenated forms of the protein determined by X-ray diffraction (Baldwin and Chothia, 1979). The initial step in the binding process involves the transition of the heme iron atom from a ferrous high-spin to a ferrous low-spin state. This transition produces a movement of the heme iron atom into the heme plane of approximately 0.5–0.6 A (Perutz, 1970, 1979). The heme iron associated proximal His residue is pulled towards the heme plane, but steric hindrance between the proximal His Ce H and the heme and the proximal His and Val (a93, b98) leads instead to a lateral motion of the proximal His across the face of the heme group. The proximal His is part of the F

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helix and so the motion of the proximal His shifts the relative position of the F helix. This in turn leads to a closing of the cavity in which Tyr (a 140, b 145) sits and favors its displacement into the surrounding solvent. As Tyr is the penultimate residue of the polypeptide chain its motion leads to a breaking of the salt bridge between the C terminal COO group of Arg a 141 or His b 146 and the NHþ 3 of Arg b 40 or Lys a 40 of the adjacent chain (Fig. 9). In this complex, mechanically amplifying process, the binding of oxygen at the heme iron of one subunit is transmitted to the adjacent subunits. The breaking of the molecular constraints on the adjacent subunit then activates that subunit, providing an enhanced affinity for the binding of the next molecule of oxygen (Perutz et al., 1987; Liddington et al., 1988). 6.2. Quantitative description of hemoglobin function The functional activity of hemoglobin has been further analyzed in quantitative terms (Monod et al., 1965; Koshland et al., 1966; Ackers et al., 1992; DiCera et al., 1987a,b; Szabo and Karplus, 1972). Although a number of models have been developed with differing degrees of complexity, the original two state or concerted model of co-operativity remains the most widely used (Monod et al., 1965); although it is recognized to have some limitations (Henry et al., 1997). The two-state model is based on two axiomatic assumptions, namely (1) that the protein exists in two quaternary states (which are viewed as equivalent to the deoxygenated and oxygenated structures determined by X-ray crystallography) which are in equilibrium under all conditions and (2) that within a particular quaternary state all of the subunits have the same affinity for oxygen. A detailed analysis of

Fig. 9. Structural motions subsequent to oxygen binding. Arrows indicate the structural motion produced subsequent to oxygen binding to the heme of the a 1 subunit of hemoglobin. The salt bridge between subunit a 1 and a 2 which is broken as a consequence of this motion is indicated by a broken line.

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such a system shows that the model fits the oxygen binding data for hemoglobin, if the low affinity quaternary structure (T state) is favored in the absence of oxygen and the high affinity (R state) dominates in the presence of saturating amounts of oxygen. Furthermore the model predicts that adult human hemoglobin will undergo a concerted conversion from the predominately T state to predominately R state on binding approximately two molecules of oxygen. This model proposes that three basic parameters govern hemoglobin function-namely KR (the oxygen affinity of the heme groups within the high affinity R structure), KT (the oxygen affinity of the heme groups within the low affinity T state structure) and L (the allosteric constant) which defines the equilibrium constant for the equilibrium between the T and R states in the absence of oxygen (Monod et al., 1965) (Fig. 10). 6.3. Heterotropic effects The binding of oxygen to hemoglobin is not only affected by the presence of oxygen but also by other small molecules called effectors. The chemical nature of effectors is very variable, ranging from Hþ , Cl , CO2 to organic phosphates. Each of these substances interacts with hemoglobin in a different way, but each action can be viewed within the two-state model of co-operativity as producing its effect by alteration of the equilibrium constant governing the distribution of the T and R state. The binding of effectors has been shown to occur preferentially to the T state and to stabilize this structure by forming additional salt bridges either within or between the various subunits (Bettati et al., 1983; Perutz, 1970; Perutz, 1989).

Fig. 10. Two-state model of the reaction of hemoglobin with oxygen. L represents the allosteric equilibrium constant and KR and KT the equilibrium constants for oxygen binding to the R and T forms of the protein. The thickness of the arrows indicates the position of the equilibrium at each step.

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The most important of the allosteric effectors of hemoglobin are protons. Protons are envisioned as producing their effect by changing the relative stabilities of the oxy R and deoxy T quaternary states. This is possible as a number of ionizing amino acids change their position within the molecule, consequent to the oxygenation linked quaternary structure change. Thus a particular pH will specifically favor a particular structural form. The outcome of this interaction is that on oxygenation hemoglobin maximally releases 2.8 protons into solution (Kilmartin et al., 1973; Kilmartin, 1974; Perutz et al., 1980). The experimental observation associated with this molecular interaction is that over the physiological pH range a drop in pH leads to a higher p50 value (lower oxygen affinity). That is, a low pH value stabilizes the low affinity T state – this is known as the Bohr effect. The amino acids which play a major role in the formation of salt bridges, affected by changes in pH, have been identified in the adult protein as residues Val a 1, His a 122, His b 2, Lys b 82, His b 143, His b 146 with His b 146, Val a 1 and His a 122 playing the major role (Ho and Russu, 1987; Kilmartin et al., 1978; Perutz et al., 1969). In quantitative terms the organic phosphates are the most significant allosteric effectors of the oxygen binding affinity of hemoglobin. In particular 2,3 diphosphoglycerate (2,3 D.P.G. or 2,3 Bis-phosphoglycerate), is produced in significant concentrations within the red blood cell in a unique side reaction of glycolysis, and is present within the red blood cell at stoichiometric ratios with hemoglobin. 2,3 D.P.G. exerts its influence on oxygen binding by preferentially binding between the b chains of hemoglobin when it is in the deoxygenated form. When hemoglobin undergoes oxygenation the b subunits move closer together and thus exclude the 2,3 D.P.G. Under physiological conditions 2,3 D.P.G. carries four negative charges which allows it to bind to the b chain inter-subunit surfaces (preferentially in the deoxygenated state) via a set of charge– charge interactions with the residues His b 2, Lys b 82, His b 143 and the b N-terminal NHþ 3 group. The concentration of 2,3 D.P.G. within the red blood cell if often altered to ameliorate patho-physiological conditions which otherwise impact on hemoglobin oxygenation. Carbon dioxide transport is also linked to the hemoglobin oxygenation process. This linkage may be direct or indirect. In the, quantitatively more significant, indirect process protons are produced within the red blood cell, by the action of intra-erythrocytic carbonic anhydrase, during the transformation of carbon dioxide to bicarbonate. These protons then lower the oxygen affinity of hemoglobin, via the action of the Bohr effect, and hence facilitate additional oxygenation of anaerobic tissues. In the direct process carbon dioxide reacts directly with the N-terminal NHþ 3 residues of each of the globin subunits of hemoglobin, particularly in the deoxygenated state, forming carbamino functions. These carbamino groups then participate in the formation of new salt bridges between the globin subunits in the deoxy T state and hence lower the oxygen affinity of hemoglobin. For adult hemoglobin, chloride ions maximally reduce oxygen affinity by a factor of seven on going from a chloride free solution to one containing 1 M chloride ions. The exact mechanism of the action of chloride ions on the oxygen affinity of hemoglobin is not certain. Despite evidence, from mutation studies, that Val 1 a and

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Lys 82 b have a significant role in the action of chloride ions as allosteric effectors of hemoglobin action (Nigen et al., 1980; Perutz et al., 1980) no X-ray evidence has ever been gathered identifying chloride ion binding to these amino acids. It is now generally accepted that the chloride ions do not bind to unique amino acid sites within the protein, but rather exert their influence by preferentially neutralizing excess positive charge within the central, water filled, cavity within the quaternary structure of the deoxygenated hemoglobin molecule (Perutz et al., 1993; Shih et al., 1993; Perutz et al., 1994; Bonaventura et al., 1994). The amino acids which provide this excess positive charge are Val a 1 (NHþ 3 ), Lys a 99, His a 103, Val b 1, His b 2, Lys b 82, Arg b 104 and His b 143. It has been proposed that the physicochemical origin of the effect of chloride ions arises from its effect on the chemical potential or activity of water itself (Colombo et al., 1994; Colombo and Bonilla-Rodriguez, 1996; Colombo and Seixas, 1999). This would then explain why there is no data available to indicate specific chloride ion binding to any of these specific sites. As a consequence of the nature of the binding of all these allosteric effectors a high concentration of the effector stabilizes the T state and hence lowers the oxygen affinity of hemoglobin. The interplay of the various allosteric effectors, which normally exert their influence in mixed solution (Antonini et al., 1965; Kilmartin and RossiBernardi, 1969; DeBruin et al., 1974; Van Beek and deBruin, 1980; Russu et al., 1982; Benesch et al., 1986; Fronticelli et al., 1984; Bucci and Fronticelli, 1985), has been quantitatively analysed using the formalism developed by Wyman (1948, 1964, 1967, 1984).

7. Embryonic hemoglobin function Although major advances in the study of embryonic gene structure, control of expression and gene switching have been made over the past twenty years a correspondingly detailed understanding of the structure and function of the embryonic hemoglobin proteins has only been made over the past 10 years. Until 1995 only two papers had appeared concerned with the function of human embryonic hemoglobins (Tuchinda et al., 1975; Huehns and Farouqui, 1975). These studies employed very small quantities of authentic human embryonic red blood cells and as a consequence many of the measurements and interpretations of embryonic function derived from these studies have proven to be of doubtful significance. However with the advent of artificial, microbial expression systems (Wagenbach et al., 1991) and more recently transgenic mice (Russell and Liebhaber, 1998; He et al., 2000; He and Russell, 2001) large quantities of the human embryonic hemoglobins have become available for structure and function studies. Studies on the human embryonic hemoglobins were made possible by the creation of plasmid-based expression systems in yeast (Wagenbach et al., 1991). These systems consist of the two appropriate globin genes, each coupled to an artificial galactose promoter sequence within a single 2 l yeast plasmid construct. By sequential growth of the transformed yeast in glucose-based media and then ethanol-based media it is possible to produce yeast cells containing high copy number for the

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plasmid, primed for heme biosynthesis, prior to induction of the globin genes. Using this method it is possible to produce yeast cultures containing 100 mg of hemoglobin per liter of culture. Extended culture schemes directed at the production of even higher yields of hemoglobin were found to invariably produce cultures containing significant levels of sulfhemoglobin (a form of hemoglobin in which the heme group is modified into a form incapable of oxygen transport under physiological conditions, produced by the introduction of a sulfur atom into the periphery of the heme group) which are not easily purified away from the native hemoglobin. Nevertheless this simple procedure has been shown to yield fully assembled and naturally functional tetrameric embryonic hemoglobins. Yeast expression systems have been developed for each of the human embryonic hemoglobins as well as adult hemoglobin. Essentially all of the structural and functional studies on the three human embryonic hemoglobins reported in the literature to date have employed embryonic hemoglobins produced in these yeast expression systems. Investigations of the basic properties of the embryonic hemoglobins produced in this way have confirmed their molecular weight, heme/globin ratio, amino acid sequence, globin composition and even post-translational modifications as being that observed in authentic samples. Adult hemoglobin produced in this way exhibits structural and functional properties indistinguishable from those of authentic samples. 7.1. Homotropic effects in human embryonic hemoglobins Under ‘‘normal’’ physiological conditions of 37 °C, pH 7.2, 100 mM chloride ions, the three human embryonic hemoglobins exhibit co-operative oxygen binding properties. Analysis of the oxygen binding curves shows that the oxygen binding process occurs with p50 values of 4, 12 and 6 mm Hg for human embryonic proteins Gower I ðf2 e2 Þ, Gower II ða2 e2 Þ and Portland ðf2 c2 Þ, respectively. These values compare with 20 and 26 mm Hg for the human fetal and adult proteins, respectively (Hellegers and Schruefer, 1961). As a group the embryonic hemoglobins exhibit somewhat lower degrees of co-operative function than the adult or fetal proteins, as expressed in terms of the Hill coefficient (h) with Hill coefficients of 1.9–2.3 as compared with 2.7 for the fetal and 2.8 for the adult proteins (Fig. 11). Further analysis of the oxygen binding curves for the embryonic hemoglobins, in terms of the two-state model of co-operativity yields values of KR , KT and L presented in Table 1. From a comparison with the equivalent parameters found for the adult protein this level of analysis suggests that the higher oxygen affinity observed for the embryonic hemoglobins arises from a lowering of the relative stability of the T state within the embryonic hemoglobins. This is evidenced by the reduction in the value of L, the allosteric equilibrium constant, which expresses the ratio of the concentrations of the T and R states, in the absence of oxygen. In contrast, the intrinsic oxygen affinities of the two quaternary states of all the hemoglobins are essentially the same as for the adult hemoglobin. The origins of this loss of stability of the T state have been traced to Val 1 a–N-Acetyl Ser 1 f; Asp 6 a–Gln 6 f; Ser 9 b– Ala 9 c, Ala 9 e; Pro 51 b–Ala 51 c and Thr b 67–Glu c 67, Lys 67 e amino acid differences which are believed to remove some of the interactions which normally

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Fig. 11. Oxygen binding to the embryonic hemoglobins. The saturation of hemoglobin with oxygen as a function of oxygen partial pressure is shown for adult hemoglobin (black circles) and embryonic hemoglobins Gower I (black inverted triangles), Gower II (open squares) and Portland (open circles).

contribute to T state stability. Furthermore the a 38 Thr–f 38 Gln change is unique in all mammalian systems to the human embryonic f chain containing hemoglobins. As detailed elsewhere within this review, this amino acid has a crucial role to play in the control of the quaternary structural change associated with the oxygenation process. Hence the presence of the f 38 Gln undoubtedly has a major significance in producing high oxygen affinity within the f chain containing human embryonic hemoglobins. However closer examination of the fit of the experimental data to the two-state model identifies a rather poor correspondence at low oxygen concentrations, where the deoxy T state of the proteins would be expected to predominate. This discrepancy can be accounted for if the two-state model is extended to allow for differences between the intrinsic oxygen affinities of the a and b type chains within the T state (a phenomenon observed in some other species). This extension provides a very good correspondence between the experimental data and the theoretical model of oxygen binding to these proteins (Zheng et al., 1999). Table 1 Two-state parameters for oxygen binding to the embryonic hemoglobins Protein

KR

KT

L

a2 b2 a2 e2 f2 e2 f2 c2

0.3 0.4 0.5 0.3

21 10 11 8

5  105 2  104 2  103 6  104

KR and KT – mm Hg; L – allosteric constant (Brittain et al., 1997).

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The oxygen affinity of the embryonic hemoglobins is significantly affected by temperature. Analysis of the temperature dependence of the oxygenation reaction, using van’t Hoff’s isochor yields values for the enthalpy of oxygenation of – 43, )51 and )42 kJ/mol for embryonic hemoglobins a2 e2 ; f2 c2 and f2 e2 , respectively (Brittain et al., 1997). These values are very similar to that reported for the adult protein (Doyle et al., 1989; Giardina et al., 1993). Thus the process of oxygen transport from mother to embryo is unlikely to be associated with any significant exchange of heat. This is in stark contrast to the situation involving fetal hemoglobin where the 30% difference in the enthalpy of oxygenation between adult and fetal hemoglobins is responsible for the dissipation of a considerable amount of heat generated by the fetal metabolism (Giardina et al., 1995). 7.2. Heterotropic effects in human embryonic hemoglobins As described above, for the adult hemoglobin, the oxygen affinity of hemoglobin can be modulated by a number of allosteric effectors. Over the physiologically appropriate pH range adult hemoglobin shows a sigmoidal dependence of oxygen affinity as a function of pH, with low affinity at low pH and an apparent transition point at pH 7.4. The fetal protein exhibits a very similar pattern of modulation by pH (Doyle et al., 1989). The embryonic proteins on the other hand show a very wide range of responses to the presence of protons in solution (Hofmann et al., 1995a). Embryonic hemoglobin Gower II ða2 e2 Þ exhibits a pattern of sensitivity towards protons which is very similar to the adult protein, but with a somewhat higher oxygen affinity at all pH values and an almost exactly similar transition value. The correspondence in general shape of the adult and Gower II Bohr effects can be rationalized by the fact that all the b chain amino acids responsible for the Bohr effect in the adult protein are present in the e chain whilst, of course, the two proteins share identical a chains. A different pattern of pH sensitivity is exhibited by embryonic hemoglobin Portland ðf2 c2 Þ with a shallower pH dependence, and a much higher general oxygen affinity than the adult hemoglobin coupled with a shift in the transition point to a pH of approximately 8.0. In this case amino acid differences occur in both the f and c chains as compared with the a and b chains. Of the amino acids identified as of functional significance in the adult protein the Val a 1 N-terminal NHþ 3 group is lost in the f chain, where it is replaced by an N-Acetyl Ser f 1. This would be expected to account for the 25% loss of Bohr effect seen in the embryonic protein (Imai, 1982; Kilmartin, 1977; O’Donnel et al., 1979). Indeed, conversion of the N-Acetyl Ser f 1 to a Val f 1 residue by means of site directed mutagenesis has been shown to restore the Bohr effect in the mutant protein to the level seen in the adult protein (Scheepens et al., 1994). The c chain also contains a His b143–Ser f143 difference which is expected to contribute to the loss of pH sensitivity seen in the embryonic protein. Embryonic hemoglobin Gower I ðf2 e2 Þ exhibits quite a different pattern of proton sensitivity. Hemoglobin Gower I shows a very high oxygen affinity at all pH values, and a shallow pH dependence, although with a transition point which is similar to

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the adult protein. The low pH sensitivity for this protein is at least in part explained by the post-translational modification of the f chain outlined above (Fig. 12). The retention of His 143 between the e and b chain almost certainly contributes to the maintained transition point. The presence of CO2 , at pH 7.4, lowers the oxygen affinity of adult hemoglobin by a factor of approximately 1.6, whilst leaving the level of co-operativity unchanged. In the case of the human embryonic hemoglobins a range of responses to the presence of CO2 is observed. Embryonic hemoglobin Gower II ða2 e2 Þ exhibits a sensitivity towards CO2 , which is essentially identical to that of the adult proteins. In contrast hemoglobin Gower I ðf2 e2 Þ shows a decrease in oxygen affinity by a factor of 1.2, whilst hemoglobin Portland ðf2 c2 Þ shows only a 10% decrease in oxygen affinity in the presence of 40 mm Hg CO2 (Hofmann et al., 1997). These marked differences in CO2 sensitivity can be understood if we consider the different ways in which hemoglobin can interact with CO2 . Within the red blood cell hemoglobin interacts with carbon dioxide in two distinct but related ways. Oxygenation of hemoglobin produces protons via the action of the Bohr effect. These protons then alter the CO2 – bicarbonate equilibrium, established by the action of carbonic anhydrase, to favor the production of CO2 . Via this indirect action of hemoglobin and CO2 , hemoglobin enhances the transport of CO2 from the tissues to the lungs. In a more direct action hemoglobin also binds CO2 directly at the N-terminal amino groups of each globin chain to produce carbamino groups. Thus, the overall interaction of hemoglobin and CO2 relates to both the strength of the Bohr effect and to the binding affinity for CO2

Fig. 12. The effect of pH on oxygen binding to the embryonic hemoglobins. The effect of pH on the binding constant for oxygen is shown for adult hemoglobin (black circles) and embryonic hemoglobins Gower I (black inverted triangles), Gower II (open squares) and Portland (open circles).

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for a particular hemoglobin. In the case of embryonic hemoglobin Gower II both the a and e globin chains share the same N-terminal amino acids as those found in the adult protein. Additionally the Bohr effect in hemoglobin Gower II is very similar to that of the adult protein. Consequently CO2 has essentially identical effects on adult and embryonic hemoglobin Gower II. In the case of the f globin chain containing embryonic hemoglobins the situation is different. The f chain is N-terminally acetylated and so incapable of combining directly with CO2 . This is expected to reduce the effect of CO2 to approximately a half, as all N-termini in the adult protein contribute equally to the CO2 binding process (Kilmartin and Rossi-Bernardi, 1973). The remaining differences between the embryonic hemoglobins Gower I and Portland and the adult protein then simply arises from the significantly different Bohr effects for each of the proteins, outlined above. The effect of 2,3 D.P.G. on the nature of the binding of oxygen to the embryonic hemoglobins also shows a pattern consistent with the amino acids sequence differences between the adult and embryonic proteins. The adult protein ða2 b2 Þ and the embryonic hemoglobins Gower II ða2 e2 Þ and Gower I ðf2 e2 Þ are all sensitive to the presence of 2,3 D.P.G., exhibiting a binding constant for the organo-phosphate of approximately 0.5 mM, although the intrinsic oxygen affinity of each protein is different (Hofmann et al., 1995a,b). This pattern can be rationalized by the observation that the charged amino acids in the b chain of the adult protein, which have been identified as the binding site for the charged organo-phosphate (see above), are all conserved in the e chains of the embryonic proteins (Fig. 13). The slightly lower binding constant observed in the case of the embryonic proteins may well arise from the Leu b 3–Phe e 3 substitution in the embryonic proteins. This substitution, although not directly affecting the charge–charge interactions responsible for D.P.G. binding, has previously been shown to lead to a slight disturbance of the stereochemistry of the 2,3 D.P.G. binding site provided by the N-terminal group of Val b 1, and as such probably acts as a second order perturbation of the binding process (Fermi and Perutz, 1981). Embryonic hemoglobin Portland ðf2 c2 Þ on the other hand shows a very much lower sensitivity towards the binding of 2,3 D.P.G. than the other proteins; reminiscent of the sensitivity of the fetal protein ða2 c2 Þ towards 2,3 D.P.G. binding (Bauer et al., 1968). Hemoglobin Portland is affected by the presence of 2,3 D.P.G. but interacts with the organo-phosphate with an equilibrium constant more than an order of magnitude higher than the other embryonic hemoglobins (Fig. 14). The c chain shows a His b 143–Ser c 143 substitution and, as such, lacks two formal charges at the 2,3 D.P.G. binding site. The c chain also has the Leu b 3–Phe c 3 substitution detailed above in the e chain. Chloride ions are also seen to have a markedly different effect on the oxygen binding characteristics of the individual embryonic hemoglobins (Hofmann et al., 1995b). As in the case of the Bohr effect, detailed above, embryonic hemoglobin Gower II ða2 e2 Þ shows a chloride ion concentration sensitivity in its oxygen binding characteristics reminiscent of, but lower than, that of the adult protein. These two proteins share a common a chain and so the difference in chloride ion sensitivity must reside in the e chain. Site directed mutation studies have identified the difference in chloride ion sensitivity as having its origins in the removal of positive charge

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Fig. 13. 2,3 D.P.G. binding to hemoglobin. The binding site between hemoglobin chain b1 (top) and b2 (bottom) is shown. The positively charged amino acids responsible for binding are indicated. 2,3 D.P.G. is shown in space fill for clarity.

Fig. 14. The effect of 2,3 D.P.G. on oxygen binding to the embryonic hemoglobins. The effect of 2,3 D.P.G. on the binding constant for oxygen is shown for adult hemoglobin (black circles) and embryonic hemoglobins Gower I (black inverted triangles), Gower II (open squares) and Portland (open circles).

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within the central water filled cavity of the embryonic hemoglobin molecule, produced by the His b 77–Asn e 77 substitution (Zheng et al., 1999). Hemoglobin Portland ðf2 c2 Þ exhibits a very weak chloride ion concentration sensitivity which can be traced to the N-terminal acetylation of the f chain and the Ser b 138–Glu f 138 substitution, both of which reduce positive charge within the central cavity. This loss of charge is compounded by the His b 143–Ser c 143 substitution, which also lowers the excess positive charge and lowers chloride sensitivity. Embryonic hemoglobin Gower I ðf2 e2 Þ is essentially devoid of any chloride ion concentration sensitivity, due to the substitutions in the f and e chains outlined above (Fig. 15). 7.3. CO toxicity The fact that hemoglobin has evolved the capacity to reversibly bind oxygen not only brings the advantage of enhanced aerobic metabolic capacity but also the potential for toxicity, induced by the binding of other substances to the heme group. In particular CO is a potentially lethal substance. CO toxicity arises in two ways. CO binds strongly to the heme iron of hemoglobin, thus preventing oxygen binding. Also, when CO partially saturates a hemoglobin molecule it renders the remaining, free, heme sites of such a high affinity for oxygen that the unbound heme sites may become ineffective in oxygen transport, over the physiologically relevant oxygen concentration range. CO in the human body originates from two basic sources.

Fig. 15. The effect of chloride ions on oxygen binding to the embryonic hemoglobins. The effect of chloride ion concentration on the binding constant for oxygen is shown for adult hemoglobin (black circles) and embryonic hemoglobins Gower I (black inverted triangles), Gower II (open squares) and Portland (open circles).

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Endogenously, CO is continually formed by the catabolism of heme. Exogenously, CO is a common environmental pollutant with many diverse origins. Free heme binds CO 103 –104 times more strongly than oxygen (Collman et al., 1976) and so would be totally inappropriate for oxygen transport at the levels of CO normally found in the body. However, when bound to protein the relative affinities of heme for CO and oxygen can be modulated by the immediate heme environment. In the case of adult hemoglobin the ratio of the CO to oxygen affinities is reduced to 250 (DiCera et al., 1987a,b). Despite continual endogenous CO production this ratio is sufficient to maintain the normal level of CO bound hemoglobin to approximately 3%, thanks to continual pulmonary gas exchange. At the fetal stage of development fetal hemoglobin is required to maintain a higher oxygen affinity than the maternal hemoglobin, so as to provide an affinity gradient for placental oxygen transfer. Although the ratio of CO to oxygen affinities for fetal hemoglobin is lowered by the fetal globin protein to a value of 180 (Longo, 1970), ambient conditions and the higher oxygen affinity of the fetal hemoglobin (p50 ¼ 20 mm Hg) are sufficient for CO bound hemoglobin levels to reach 11% in utero (Hill et al., 1977). At the embryonic stage of development, the necessity for very much higher oxygen affinity means that in order to avoid CO toxicity, the embryonic hemoglobins must exhibit enhanced discrimination between CO and oxygen binding. Recent investigations show that all the human embryonic hemoglobins have ratios of CO affinity to oxygen affinity of only 85 (Hofmann and Brittain, 1998) – approaching the value 30 found for myoglobin (Rossi-Fanelli and Antonini, 1958), which for similar reasons must have significant discriminatory power. The origin of the discriminatory power of heme proteins in selection against CO binding has only been investigated in detail in the case of myoglobin (Springer et al., 1989; Traylor and Berzinis, 1980; Sigfridsson and Ryde, 1999; Lim et al., 1997; Kachalova et al., 1999; Slebodnick and Ibers, 1997). However, it is likely that similar factors and mechanisms are the origin of the high level of discrimination observed for the embryonic hemoglobins. In the case of myoglobin three contributing factors have been identified as significant; (1) Steric hindrance of the formation of linear bound diatomic molecules by virtue of the presence of the distal His residue is a major discriminator against CO binding, and has been shown to reduce CO affinity by two orders of magnitude in model compounds (Collman et al., 1983). (2) Hydrogen bonding, of bound oxygen by the distal His residue, favors oxygen binding by as much as a magnitude in model compounds (Olson et al., 1988; Lukin et al., 2000). (3) The polarity of the heme pocket stabilizes bound oxygen and can alter oxygen affinity by a factor of 40 in model systems (Traylor et al., 1984; Traylor et al., 1985; Lavalette et al., 1984). Identification of the relative significance of these factors in the case of the human embryonic hemoglobins must await a detailed analysis of the appropriate protein structures. 7.4. Redox activity in embryonic hemoglobins In order for hemoglobin to perform its normal physiological role of oxygen transporter it is necessary for the heme iron atom to remain in the ferrous state.

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However, during the normal oxygenation/deoxygenation cycle the oxygenated form of the protein, which is transiently formed, is metastable towards oxidation. The oxidation process itself occurs by two main mechanisms; (1) anion induced oxidation (Wallace et al., 1974, 1982) and (2) spontaneous monomolecular decomposition to form superoxide (Weiss, 1964). Of these two mechanisms the anion induced mechanism is the most studied and best characterized. Many common anions are effective agents for inducing hemoglobin oxidation (Antonini and Brunori, 1971; Mansouri and Winterhalter, 1973). Despite its low nucleophilicity, the high concentration of chloride ions in red blood cells means that chloride is probably quantitatively the most important anion responsible for hemoglobin oxidation (Wallace et al., 1974). The embryonic hemoglobins undergo anion induced oxidation in the presence of chloride ions, but at rates which are considerably slower than those observed for the adult protein (Robson and Brittain, 1996a). However, all the human hemoglobins exhibit essentially identical pH dependences for the chloride induced oxidation process, which has been attributed to protonation of bound superoxide, prior to dissociation from the heme iron atom. In the case of the adult the sum of the oxidation processes leads to the oxidation of approximately 3% of the total circulatory hemoglobin each day (Jaffe, 1964). Unfortunately data is not available for the level of oxidation in vivo for embryonic hemoglobins. Nevertheless the fact that embryonic red blood cells, unlike adult red blood cells, are metabolically fully active means that it is probable that embryonic hemoglobins are oxidized to an appreciable extent each day. Under normal circumstances the oxidized hemoglobin formed within the adult red blood cell is re-reduced to the active ferrous form. This re-reduction process involves the transfer of electrons from intracellular NADPH, via a flavoprotein enzyme, met-hemoglobin reductase, to cytochrome b5 and then to oxidized (met) hemoglobin (Hultquist et al., 1974; Passon and Hultquist, 1972). Until recently the mechanism of this re-reduction process was not well characterized. Brownian dynamic simulation (Northrup et al., 1987, 1988; Eltis et al., 1991) of the reaction between cytochrome b5 and oxidized hemoglobins has highlighted a number of interesting aspects of this protein–protein interaction (Kidd et al., 2002). It is clear that the electric charge on both molecules has a significant role. Cytochrome b5 possesses a ring of negatively charged amino acids around its heme group, which docks with a ring of positively charged amino acids surrounding the hemoglobins heme groups. This then leads to an efficient transfer of electrons from the cytochrome b5 to the oxidized hemoglobin, restoring it to its normal physiological function. These theoretical calculations have been verified using rapid mixing experiments to follow the reduction process (Brittain et al., 2002). Both theoretical and experimental investigations of this reaction have highlighted the effect of amino acid differences between various hemoglobins on the efficiency of the re-reduction process. In particular, it has been found that the embryonic hemoglobins exhibit rates of re-reduction which range from a magnitude lower than the a or b globins, in the case of f globin, to two orders of magnitude greater, in the case of e globin. In all cases, calculations indicate that, at the concentrations present, re-reduction of the embryonic hemoglobins would be an efficient process, in vivo.

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7.5. Heme binding and dimerization NMR studies on recombinant embryonic hemoglobins indicate that the embryonic globins bind heme rapidly, to directly achieve the equilibrium rotamer distribution (Mathews and Brittain, 2001). Furthermore, exchange studies indicate that the embryonic hemoglobins bind heme over a magnitude more tightly than does the adult protein even though the proteins show essentially identical conformational flexibility (Robson and Brittain, 1996a,b). At pH 9.0 both adult and embryonic hemoglobins all dissociate into dimers with associated equilibrium constants in the micro-molar range (Robson and Brittain, 1996a). However, at low pH, embryonic hemoglobin Portland in particular shows a very high degree of globin dissociation to the point where globin chain shuffling takes place to produce measurable levels c4 tetramers in equilibrium with dimeric and monomeric globin chains (Kidd et al., 2001a–c). 7.6. Kinetic studies Whilst equilibrium measurements have traditionally been used to study physiological function, in order to make distinctions between mechanistic models and for comparative purposes, they are necessarily limited by the fact that all equilibrium constants are the ratio of kinetic constants. Hence, determination of kinetic parameters holds the possibility of gaining further insight into molecular action and refinement of mechanistic models. Unfortunately the rates of reaction of hemoglobin with many of the substances of interest are very high and so specialized equipment is required to make the appropriate measurements in the ls to ms time range. 7.6.1. Ligand binding The rate of oxygen dissociation from hemoglobin is one of the easier rates to measure as it is rather low in most cases. The rate of oxygen dissociation is usually obtained by displacement with a higher affinity ligand, such as carbon monoxide or else by rapid mixing with a deoxygenating substance such as sodium dithionite. In these circumstances the rate observed corresponds to the rate of oxygen dissociation from the oxy R state of the protein and is normally monitored using absorption spectroscopy. In the case of human adult hemoglobin rapid deoxygenation of a solution containing oxyhemoglobin produces a time course for the deoxygenation reaction in the hundreds of millisecond time range consistent with a single process in the absence of chloride ions and the presence of two processes, which have been assigned to deoxygenation of the a and b chain, in the presence of 100 mM chloride ions. The adult protein shows a marked pH dependence of the rate of oxygen dissociation, mirroring the alkaline Bohr effect. The human embryonic hemoglobins show a similar pattern of oxygen dissociation, but with the f chains losing oxygen at approximately twice the rate of the a chains (see Table 2). The rates of oxygen dissociation from the f chain containing embryonic proteins also exhibit a much lower sensitivity towards pH, in line with their much lower Bohr effects.

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Oxygen association rates can be measured in the micro-second time range, with some difficulty, by following oxygen recombination to partially photolyzed, oxygenated hemoglobin. If the level of photolysis of the oxyhemoglobin is kept to less than 5% then recombination occurs onto oxy R state hemoglobin (Hopfield et al., 1971). The same pattern of reactivity as seen in the dissociation experiments, is seen in the association reactions, with recombination more obviously consisting of the sum of two separate reactions in the presence of 100 mM chloride ions. However, whereas the b chains of the adult protein dissociate oxygen at a slower rate than the embryonic chains, the adult b chains recombine with oxygen at a faster rate than do the embryonic chains. On the other hand the a chains and the embryonic f chain show essentially identical dissociation rates and closely similar association rates. Interestingly the ratio of the association and dissociation rates for each chain yield values for the R state equilibrium constant identical, within experimental error, to that determined in equilibrium experiments. Furthermore the value obtained for the R state equilibrium constant is essentially independent of the protein studied (Table 2). These data lead to the conclusion that the oxygen binding affinity differences between the adult and embryonic hemoglobins, outlined above, do not arise from intrinsic differences in the oxygen binding properties of the R states of the proteins. Although it is extremely difficult to obtain convincing T state rates for oxygen binding to hemoglobin it is possible to obtain rate data for the quaternary structure change associated with oxygen binding. Full photolysis of the carbon monoxide derivative of hemoglobin yields initially unliganded R state hemoglobin. Unlike oxygen, carbon monoxide reacts rather slowly with hemoglobin and so it is possible to observe the transition from unliganded R state hemoglobin to deoxy T state hemoglobin prior to recombination with carbon monoxide. Such experiments show that the rate of the R–T state transition is essentially independent of the hemoglobin studied. Remembering however, that the embryonic hemoglobins exhibit values of the allosteric equilibrium constant (L) which are lower than that shown by the adult protein and that L is the ratio of the R–T rate divided by the T–R rate, it is apparent that the relative loss of stability of the T state within the embryonic proteins arises from an easier T–R transition (Zheng et al., 1999).

Table 2 Rates of reaction of oxygen with embryonic hemoglobins Protein

on

a2 b2 a2 e2 f2 e2 f2 c2

40, 49, 70, 54,

kR 12 11 11 11

off

kR

140, 94, 94, 92,

42 28 24 38

R!T

kin

1.9 2.5 2.9 2.6

0.5 0.9 1.0 0.8

KR

kR – lM1 s1 ; off kR – s1 ; R ! T s1 ð104 Þ. KR – mm Hg (cf. Table 1 equilibrium constant, measured under slightly different conditions) (Brittain et al., 1997).

on

kin

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7.6.2. Subunit dissociation Kinetic measurements are also useful in determining the strength of inter-subunit contacts in both the oxygenated and deoxygenated forms of the protein. Photolysis of the carbon monoxide derivative of hemoglobin at low pH yields values for the dimerization of both adult and embryonic liganded hemoglobins in the low lM range, as do indirect measurements of dissociation obtained from studies of heme exchange reactions (Robson and Brittain, 1996a,b; Hofmann and Brittain, 1996). Equilibrium measurements of the dissociation of the deoxy form of hemoglobin are more difficult as the protein dissociates only at very low concentrations. However, using rapid mixing of oxygenated hemoglobin with sodium dithionite, and following the reaction at the appropriate wavelength, over a range of protein concentrations, it has been shown that the rate of conversion of deoxy dimers to tetramers is very similar for the adult and embryonic hemoglobins (Kellet and Gutfreund, 1970; Hofmann and Brittain, 1996). The opposing reaction can be measured by rapidly mixing reduced hemoglobin with haptoglobin, which binds dimeric hemoglobin, produced by dissociation of the deoxy protein (Nagel and Gibson, 1971; Chiancone et al., 1968) and following the subsequent reaction. Such studies indicate that adult hemoglobin and the e chain containing embryonic hemoglobin have similar rates of dimerization. The embryonic f2 c2 hemoglobin, on the other hand, has a significantly lower rate of dissociation. The ratios of these rate data indicate that the equilibrium constant for dissociation of the deoxy form of the a chain containing adult and embryonic hemoglobin Gower II is more than a magnitude smaller than that of the f chain containing embryonic hemoglobins (Hofmann and Brittain, 1996) (Table 3).

8. Embryonic hemoglobin structure Although adult hemoglobin was one of the first proteins ever to be crystallized and its structure solved, the embryonic hemoglobins have proven to be extremely refractory towards crystallization. To date embryonic hemoglobin Gower II ða2 e2 Þ and Portland ðf2 c2 Þ have been crystallized, but only the Gower II structure has been solved using X-ray diffraction. In order to circumvent these problems the embryonic globin structures have been solved within the context of other protein tetramers.

Table 3 Subunit dissociation of embryonic hemoglobins Protein

Oxy Kd ðlMÞa

Oxy Kd ðlMÞb

Deoxy Kd c

a2 b2 a2 e2 f2 e2 f2 c2

2.0 2.5 2.0 3.4

1.5 1.0 1.5 4.0

6.6 8.3 13 50

a

Kd – from kinetic measurements (Hofmann and Brittain, 1996). Kd – from equilibrium measurements (Robson and Brittain, 1996a,b). c Kd – 109 M. b

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8.1. Embryonic hemoglobin Gower II structure Embryonic hemoglobin Gower II was crystallized at pH 8.5 in the carbon monoxide form and its structure solved by X-ray diffraction to 2.9 A resolution, and the structure refined to an R factor of 18.6% (Sutherland-Smith et al., 1998) (Fig. 16). The quaternary structure of the carbon monoxide form of hemoglobin Gower II lies between that reported for the R and R2 states, lying closer to the R2 structure (Silva et al., 1992; Smith et al., 1991; Smith and Simmons, 1994). The tertiary structure of the a subunit of hemoglobin Gower II is essentially identical to that of the a chain in the adult hemoglobin (Shaanan, 1983). The largest conformational difference is observed in the displacement of the C-terminal region of the loop between the C and E helices with a shift of 1.2 A of residue 50, although this region of the molecule as a whole is quite flexible. The structure of the heme pocket of the a subunit of Gower II is, within experimental error, identical to that reported for the adult protein (Derewenda et al., 1990). The tertiary structure of the e subunit is similar to that reported for the b chain. The most significant difference between the e and b chain is a shift of the N-terminus and the A helix (Katz et al., 1994). The N-terminal region of the e chain contains a

Fig. 16. The structure of embryonic hemoglobin Gower II ða2 e2 Þ.

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large number of amino acid substitutions relative to the b chain and many of these amino acids have altered interactions. In particular, the altered conformation in the N-terminal and A helix region are stabilized by additional hydrogen bonding between the carbonyl of His 2 and NZ of Lys 132 in the e chain. A number of amino acid substitutions between the e and b chains are located within the internal core of the molecule, but appear to have no major structural effects. The overall structure of the e chain heme pocket is essentially identical to that of the b chain. There is, however, a single amino acid substitution within the heme pocket, namely 70 b Ala–e Ser. There is an 83 b Gly–e Pro substitution at the beginning of the F helix which may well reduce the flexibility of the EF loop and hence contribute to the destabilization of the T state and hence the rise in oxygen affinity of hemoglobin Gower II. In the e chain Lys 66 makes an interaction with one of the heme propionate side chains and this may well account for the higher heme affinity observed in this globin (Robson and Brittain, 1996a,b). The a1 e1 interface in Gower II contains three amino acid substitutions compared with the a2 b1 interface. The b 55 Met–e 55 Leu substitution leads to the loss of a hydrophobic contact, but this is replaced by the b 112 Cys–e 112 Ile substitution which makes a new non-polar contact with a 106. The b 116 His–e 116 Thr substitution results in the loss of a hydrogen bond at the interface. The a1 b2 interface is strongly conserved and only a single b 43 Glu–e 43 Asp substitution occurs at this interface. However e 43 Asp makes an ionic interaction with a 92 Arg in a similar manner to that of b 43 Glu resulting in little overall change. 8.2. The structure of c globin subunits Embryonic hemoglobin Portland has been crystallized but the asymmetric unit contains eight tetrameric hemoglobin molecules and the solution of the protein structure has proven intractable. However, during crystallization trials it was found that at low pH samples of hemoglobin Portland yielded crystals of hemoglobin Barts ðc4 Þ by subunit reorganization (Kidd et al., 2001a,b). The structure of hemoglobin Barts has been solved at high resolution (1.7 A) in both the azide and carbon monoxide forms, thus leading to a detailed structure of the embryonic c globin chain. The c globin shows a similar structure to the b chain with a closer correspondence to the b structure in Hemoglobin H ðb4 Þ (Borgstahl et al., 1994) than that in hemoglobin A ða2 b2 Þ. The major difference between the c and b globin tertiary structure is in the N-terminal region of the protein. The A helix is tilted towards the E helix so that the N-terminus is displaced by 2 A. This movement is propagated along the peptide chain until the N-terminal Gly residue is displaced by 4 A from its position in the b chain. Such a motion has previously been reported for the deoxy fetal hemoglobin (Frier and Perutz, 1977) and is implicated in the greater tetramer stability reported for hemoglobin F relative to adult hemoglobin (Manning et al., 1999; Dumoulin et al., 1997). The efficient assembly of c chains into dimers and tetramers, as compared to the monomer–tetramer equilibrium of the b chains, has been traced to the b 116 His–c 116 Ile substitution (Kidd et al., 2001a). The analysis of the structure of hemoglobin Barts and its comparison with that of hemoglobin H

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has identified the common Arg 40/Asp 99 interactions as a major contributor to the fact that the b type chains readily form homotetramers whilst a chains do not. 8.3. f globin structure In the absence of either f2 e2 or f2 c2 structures the structure of the f chain has been determined within the context of the structure of a chimeric fðhumanÞ2 bðmouseÞ2 protein (Kidd et al., 2001c). Using transgenic technologies it is possible to produce chimeric embryonic human/adult mouse hemoglobins in large quantities. Crystals of fðhumanÞ2 bðmouseÞ2 hemoglobin have been grown at pH 7.4 in the presence of carbon monoxide. The structure of the mouse b chain is essentially identical to that of the human b chain in adult human hemoglobin. The f chain however shows significant structural differences from the human a chain. The C-terminal Ca atoms diverge by 9A. The heme pockets of human a and human f chains are very similar. f Lys 61 forms an interaction with the heme propionate side chain reminiscent of the interaction seen with b Lys 66. The f1 b1 interface contains two amino acid substitutions relative to the a1 b1 interface. However, the interactions disrupted in the a1 b2 interface are essentially compensated for by new interactions in the f1 b1 interface. The weaker a1 b2 interface is altered in the f1 b2 interface by a single substitution in the f chain. This change is the a(human) 38 Thr–f 38 Gln substitution alluded to above. Mutation studies on the adult human protein have identified this Thr–Gln substitution as having a significant role in the propagation of high oxygen affinity in f containing hemoglobins by altering the nature of the sliding contact involved in the T–R transition (Zheng et al., 1999).

9. Summary A series of gene duplication events has led from myoglobin to the creation of five different globin proteins, which are employed for oxygen transport in humans. These proteins combine to produce five different hemoglobin molecules. The genes encoding the globin proteins reside on chromosomes 11 and 16 in quite different chromatin environments. Expression of these proteins, in a temporally co-ordinated manner, is controlled primarily by upstream control elements which sequentially interact with the series of globin genes in a process which includes both autonomous and competitive interactions, mediated by a large range of transacting factors. This complex process leads to the production of three classes of hemoglobin molecules optimized to function during the embryonic, fetal and adult phases of development. The embryonic hemoglobin class contains three molecules which function prior to the establishment of a functional placenta. These hemoglobins predominate in the circulation from week four of gestation until approximately week 12, supporting oxygen transport from the maternal interstitial fluid to the rapidly developing embryo. Although the time period over which these hemoglobins function is small it represents the most rapidly changing physiological/developmental period of human life. As a consequence of these rapid changes the embryonic hemoglobins need to

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fulfill a wide range of oxygen delivery demands, and as such do not form a homogeneous group. Indeed, functional studies identify a wide range of both intrinsic oxygen affinity and sensitivity towards allosteric modulation within the embryonic hemoglobins. The earliest embryonic hemoglobin exhibits high affinity oxygen binding characteristics coupled to a very low sensitivity towards modulation of oxygen affinity, reminiscent of the functioning of myoglobin. The intermediate embryonic hemoglobin exhibits somewhat lower oxygen affinity and a greater degree of allosteric modulation. The final embryonic hemoglobin, which is expressed in the time frame which covers the embryonic to fetal transition, shows properties quite similar to the fetal hemoglobin with moderately higher oxygen affinity coupled to an allosteric sensitivity which closely matches that of the fetal and adult proteins. The sequential, overlapping expression of the three embryonic hemoglobins thus provides oxygen transport properties spanning myoglobin like properties to that of full allosteric modulation, and consequently provide for the requirements ranging from vitelline to placental circulations. Structural and functional data have begun to appear, which are beginning to identify the molecular origins of the embryonic hemoglobin function. However, much work remains to clarify the exact origins of the variety of functional behavior expressed within the embryonic hemoglobins. Such knowledge will not only provide a full molecular description of the working of the embryonic hemoglobins, but will undoubtedly contribute to our wider understanding of hemoglobin function in general and also the origins of a number of hemoglobinopathies.

Acknowledgements I would like to acknowledge my indebtedness to the graduate students and postdoctoral workers in my laboratory who contributed so significantly to our understanding of these molecules. I also acknowledge Prof. R. Wells (Auckland, New Zealand), Prof. E.N. Baker (Auckland, New Zealand), Prof. R. Weber (Aarhus, Denmark), Dr. N. Watmough (East Anglia, U.K.) and Dr. J. E. Russell (Philadelphia, U.S.A.) for their invaluable collaborations. All molecular structures were prepared using PYMOL, DeLano Scientific, San Carlos, CA, USA. http://www. pymol.org. Thanks to Dr. J. M. Brittain and Dr. A. Mathews for proof reading the manuscript.

References Abraham, D.J., Kellog, G.E., Holt, J.M., Ackers, G.K., 1997. Hydropathic analysis of the non-covalent interactions between molecular subunits of structurally characterized hemoglobins. J. Mol. Biol. 272, 613–632. Ackers, G.K., Doyle, M.L., Myers, D., Daugherty, M.A., 1992. Molecular code for cooperativity in hemoglobin. Science 255, 54–62.

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

331

Albitar, M., Cash, F.E., Peschle, C., Liebhaber, S.A., 1992. Developmental switch in the relative expression of a1 and a2 globin genes in humans and in transgenic mice. Blood 79, 2471–2474. Andrews, N.C., Erdjument-Bromage, H., Davidson, M.B., Tempst, P., Orkin, S.H., 1993. Erythroid transcription factor NF-E2 is a hemopoietic-specific basic-leucine zipper protein. Nature 362, 722–728. Antonini, E., Brunori, M., 1971. In: Hemoglobin and Myoglobin in Their Reactions with Ligands. NorthHolland, Amsterdam, Netherlands, pp. 20–52. Antonini, E., Wyman, J., Brunori, M., Fronticelli, C., Bucci, E., Rossi-Fanelli, A., 1965. Studies on the relations between molecular and functional properties of hemoglobin. The influence of temperature an the Bohr effect in human and horse hemoglobin. J. Biol. Chem. 240, 1096–1103. Armstrong, J.A., Bieker, J.J., Emerson, B.M., 1998. A SW1/SNF-related chromatin remodeling complex, E-RC1, is required for tissue – specific transcriptional regulation by EKLF in vitro. Cell 95, 93–104. Aschauer, H., Schafer, W., Sanguansermsri, T., Braunitzer, G., 1981. Human embryonic hemoglobins; Acetylserylleucyl-threonine is the amino terminal sequence of zeta-chains. Hoppe-Seyler’s Z. Physiol. Chem. 362, 1657–1659. Baldwin, J., 1975. Structure and function of hemoglobin. Prog. Biophys. Mol. Biol. 29, 225–320. Baldwin, J., Chothia, C., 1979. Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism. J. Mol. Biol. 129, 175–220. Bauer, C., Ludwig, I., Ludwig, M., 1968. Different effects of 2,3 diphosphoglycerate and adenosine triphosphate on the oxygen affinity of adult and foetal human haemoglobin. Life Sci. 7, 1336–1343. Behringer, R.B., Hammer, R.E., Brinster, B.L., Palmiter, R.D., Townes, T.M., 1987. Two 30 sequences direct adult erythroid-specific expression of human b-globin genes in transgenic mice. Proc. Natl. Acad. Sci. USA 84, 7056–7060. Benesch, R.E., Benesch, R., Kwong, S., McCord, J.M., 1986. Binding of diphosphoglycerate and ATP to oxyhemoglobin dimmers. J. Mol. Biol. 190, 481–486. Bernstein, P., Peltz, S.W., Ross, J., 1989. The poly(A)–poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. Mol. Cell. Biol. 9, 659–670. Bettati, S., Mozzarelli, A., Perutz, M.F., 1983. Allosteric mechanism of hemoglobin: rupture of salt bridges raises oxygen affinity of the T-structure. J. Mol. Biol. 281, 581–585. Blake, C.C.F., 1981. Exons and the structure, function and evolution of hemoglobin. Nature 291, 616. Bloom, W., Bartelmez, G.W., 1940. Hematopoiesis in young human embryos. Am J. Anat. 67, 21–49. Bonaventura, C., Arumugam, M., Cashon, R., Bonaventura, J., Moo-Penn, W.F., 1994. Chloride masks effects of opposing positive charges in HbA and Hb Hinsdale (b 139 Asn–Lys) that can modulate cooperativity as well as oxygen affinity. J. Mol. Biol. 239, 561–568. Borgstahl, G.E.O., Rogers, P.H., Arnone, A., 1994. The 1.8 A structure of carbonmonoxy-b4 hemoglobin. Analysis of a homotetramer with the R quaternary structure of liganded a2 b2 hemoglobin. J. Mol. Biol. 336, 817–830. Boyer, S.H., Belding, T.K., Margolet, K.L., Noyes, A.N., 1975. Fetal hemoglobin restriction to a few erythrocytes (F-cells) in normal human adults. Science 188, 361–363. Brittain, T., Simpson, R., 1989. Stopped-flow kinetics of oxygen uptake and release by embryonic red blood cells. Biochem. J. 261, 1009–1013. Brittain, T., Hofmann, O.M., Watmough, N.J., Greenwood, C., Weber, R.E., 1997. A two state analysis of cooperative oxygen binding in the three human embryonic haemoglobins. Biochem. J. 326, 299–303. Brittain, T., Kidd, R.D., Baker, E.N., 2002. Electron transfer between cytochrome b5 and some oxidized haemoglobins the role of ionic strength. J. Inorg. Biochem. 88, 328–334. Brown, K.E., Amoils, S., Horn, J.M., Buckle, V.J., Higgs, D.R., Merkenschlager, M., Fisher, A.G., 2001. Expression of a and b globin genes occurs within different nuclear domains in hemopoietic cells. Nat. Cell. Biol. 3, 602–606. Brownell, J.E., Zhou, J., Ranelli, T., Kobayashi, R., Edmondson, D.K., Roth, S.Y., Allis, C.D., 1996. Tetrahymena histone acetyl-transferase A: A homolog to yeast Gen 5p linking histone acetylation to gene activation. Cell 84, 843–851. Bucci, E., Fronticelli, C., 1985. Anion Bohr effect of human hemoglobin. Biochemistry 24, 371–376.

332

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

Bungert, J., Dave, U., Lim, K.C., Kieuw, K.H., Shavit, J.A., Liu, Q., Engel, J.D., 1995. Synergistic regulation of human b globin gene switching by locus control region element HS3 and HS4. Genes Dev. 9, 3083–3096. Cairns, L.A., Crotta, S., Minuzzo, M., Moroni, E., Granucci, F., Nicolis, S., Schuro, R., Pozzi, L., Giglioni, B., Riccardi-Castagnoli, L., Ottolenghi, S., 1994. Immortalisation of multipotent growthfactor dependent hemopoietic progenitors from mice transgenic for GATA-1 driven SV40 tsA58 gene. EMBO J. 13, 4577–4586. Chan, J.H., Han, X.-L., Kan, J.W., 1993. Cloning Nrf1 and NF-E2 related transcription factor, by genetic selection in yeast. Proc. Natl. Acad. Sci. USA 90, 11371–11375. Charache, S., Terrin, M.L., Moore, R.D., 1995. Effects of hydroxyurea on the frequency of painful crises in sickle cell anaemia. New. Engl. J. Med. 332, 1317–1322. Chen, J.J., London, I.M., 1995. Regulation of protein synthesis by heme regulated eIF-2a kinase. TIBS 20, 105–108. Chiancone, E., Alfsen, A., Ioppolo, C., Vecchini, P., Finazzi-Agro, A., Wyman, J., Antonini, E., 1968. Studies on the reaction of haptoglobin with hemoglobin and hemoglobin chains. J. Mol. Biol. 34, 347– 356. Clegg, J.B., Gagnon, J., 1981. Structure of the zeta chain of human embryonic hemoglobin. Proc. Natl. Acad. Sci. USA 78, 6076–6080. Collman, J.P., Brauman, J.I., Halbert, T.R., Suslik, K.S., 1976. Nature of oxygen and carbon monoxide binding to metalloprphyrins and heme proteins. Proc. Natl. Acad. Sci. USA 73, 3333–3337. Collman, J.P., Brauman, J.I., Iverson, B.L., Sessler, J.L., Morris, R.N., Gibson, Q.H., 1983. Oxygen and carbon monoxide binding to iron (II) porphyrins. A comparison of the ‘‘picket fence’’ and ‘‘pocket’’ porphyrins. J. Am. Chem. Soc. 105, 3052–3064. Colombo, M.F., Bonilla-Rodriguez, G.O., 1996. The water effect on allosteric regulation of hemoglobin probed in water/glucose and water/glycine solutions. J. Biol. Chem. 271, 4895–4899. Colombo, M.F., Seixas, F.A.V., 1999. Novel allosteric conformation of human Hb revealed by the hydration and anion effects on O2 binding. Biochemistry 38, 11741–11748. Colombo, M.F., Rau, D.C., Parsegian, V.A., 1994. Reevaluation of chloride regulation of hemoglobin oxygen uptake: the neglected contribution of protein hydration in allosterism. Proc. Natl. Acad. Sci. USA 91, 10517–10520. Cote, J., Quinn, J., Workman, J.L., Peterson, C.L., 1994. Stimulation of GAL 4 derivative binding to nucleosomal DNA by the yeast SW1/SNF complex. Science 265, 53–60. Courney, A.J., Tijian, R., 1998. Analysis of SP1 in vivo reveals multiple transcription domains including a novel glutamine-rich activation motif. Cell 55, 887–898. Craddock, C.F., Vyas, P., Sharpe, J.A., Ayyub, H., Wood, W.G., Higgs, D.R., 1995. Contrasting effects of alpha and beta globin regulatory elements on chromatin structure may be related to their different chromosomal environments. EMBO J. 14, 1718–1726. Craiks, S., Buchman, S.R., Beychok, S., 1981. O2 binding properties of the product of the central exon of b globin gene. Nature 291, 87–90. Crossley, M., Orkin, S.H., 1994. Phosphorylation of the erythroid transcription factor GATA-1. J. Biol. Chem. 269, 16589–16596. Crossley, M., Tsang, A.P., Bicker, J.S., Orkin, S.H., 1994. Regulation of the erythroid Kruppel-like factor (EKLF) gene promoter by the erythroid transcription factor GATA-1. J. Biol. Chem. 269, 15440– 15444. DeBenedetti, A., Baglioni, C., 1983. Phosphorylation of initiation factor eIF-2alpha, binding of mRNA to 48S complexes and its reutilization in initiation of protein synthesis. J. Biol. Chem. 258, 14556– 14562. DeBruin, S.H., Rollema, H.S., Jannsen, J.L.H.M., vanOs, G.A.J., 1974. The interaction of 2,3 diphosphoglycerate with human deoxy and oxy hemoglobin. Biochem. Biophys. Res. Commun. 58, 210–215. Derewenda, Z., Dodson, G., Emsley, P., Harris, D., Nagai, K., Perutz, M.F., Reynaud, J., 1990. Stereochemistry of carbon monoxide binding to normal human adult and Cowtown haemoglobins. J. Mol. Biol. 211, 515–519.

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

333

DeSimone, J., Heller, P., Hall, L., Zwiers, 1982. 5-Azacytidine stimulates fetal hemoglobin synthesis in anaemic baboons. Proc. Natl. Acad. Sci. USA 79, 4428–4431. DiCera, E., Doyle, M.L., Connelly, P.R., Gill, S.J., 1987a. Carbon monoxide binding to human hemoglobin Ao . Biochemistry 26, 6494–6502. DiCera, E., Roberts, C.H., Gill, S.J., 1987b. Allosteric interpretation of the oxygen binding reaction of human hemoglobin tetramers. Biochemistry 26, 4003–4008. Dickerson, R.E., 1971. The structure of cytochrome c and the rate of molecular evolution. J. Mol. Evol. 1, 26–45. Dickerson, R.E., Geis, I., 1983. Hemoglobin: Structure, Function, Evolution and Pathology. Benjamin Cummings, California, USA. Diesseroth, A., Nienhuis, A., Turner, P., Valez, R., Anderson, W.F., Ruddle, F.H., Lawrence, J., Greagan, R., Kucherlapti, R., 1977. Localisation of human alpha globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay. Cell 12, 205–218. Diesseroth, A., Nienhuis, A., Lawrence, J., Gillies, R., Turner, P., Ruddle, F.H., 1978. Chromosomal localization of human beta globin gene on chromosome 11 in somatic cell hybrids. Proc. Natl. Acad. Sci. USA 75, 145–1460. Dillon, N., Grosveld, F., 1991. Human c-globin genes silenced independently of other genes in the betaglobin locus. Nature 350, 252–254. Donze, D., Townes, T.M., Bieker, J.S., 1995. Role of erythroid Kruppel-like factor in human c and b globin gene switching. J. Biol. Chem. 270, 1955–1959. Doyle, M.L., Gill, S.J., DeCristofaro, R., Castagnola, M., DiCera, E., 1989. Temperature and pH dependence of the oxygen binding reaction of human foetal haemoglobin. Biochem. J. 260, 617–619. Doyle, M.L., Lew, G., Turner, G.J., Ruchnagel, D., Ackers, G.K., 1992. Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin Ypsilanti represent an allosteric intermediate? Proteins 14, 351–362. Dumoulin, A., Manning, L.R., Jenkins, W.I., Winslow, R.M., Manning, J.M., 1997. Exchange of subunit interfaces between recombinant adult and fetal hemoglobins. J. Biol. Chem. 272, 31326–31332. Eaton, W.A., 1980. The relationship between coding sequences and function in hemoglobin. Nature 284, 183–185. Efstratiadis, A., Posakony, J.W., Maniatis, T., Lawn, R.M., O’Connell, C., Spitz, R.A., DeRiel, J.K., Forget, B.G., Weissman, S.M., Slighton, J.L., Blechl, A.E., Smithies, O., Baralle, F.E., Shoulders, C.C., Proudfoot, N.J., 1980. The structure and evolution of the human beta-globin gene family. Cell 21, 653–668. Ellis, J., Talbot, D., Dillon, N., Grosveld, F., 1993. Synthetic human b-globin 50 HS2 constructs function and locus control regions only in multicopy transgene concatamers. EMBO J. 12, 127–134. Ellis, J., Tan-Un, K.C., Harper, A., 1996. A dominant chromatin opening activity in 50 hypersensitive site 3 of the human b-globin locus control region. EMBO J. 15, 562–568. Eltis, L.D., Herbert, R.E., Barker, P.D., Mauk, A.G., Northrup, S.H., 1991. Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5 – experimental and theoretical analysis. Biochemistry 30, 3663–3674. Engel, J.D., Tanimoto, K., 2000. Looping, linking and chromatin activity. Cell 100, 499–502. Fantoni, A., Farace, M.G., Gambari, L., 1981. Embryonic hemoglobins in man and other mammals. Blood. 57, 623–633. Fermi, G., Perutz, M.F., 1981. Haemoglobin and myoglobin. In: Phillips, D.C., Richards, F.M. (Eds.), Atlas of Molecular Structures in Biology. Clarendon Press, Oxford, UK. Fermi, G., Perutz, M.F., Shaanan, B., Fourme, R., 1984. The crystal structure of deoxyhaemoglobin at 1.7 A resolution. J. Mol. Biol. 175, 159–174. Filipe, A., Li, Q., Devereaux, S., Godin, I., Romeo, P.H., Stamatoyannopoulos, G., Mignotte, V., 1999. Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching. EMBO J. 18, 687–697. Flint, J., Thomas, K., Micklem, G., Raynham, H., Clark, K., Doggett, N.A., King, A., Higgs, D.R., 1997. The relationship between chromosome structure and function at a human teleomeric region. Nat. Genet. 15, 252–257.

334

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

Ford, L.P., Bagga, P.S., Wiluzz, J., 1997. The poly (A) tail inhibits the assembly of a 30 to 50 exonuclease in an in vitro RNA stability system. Mol. Cell. Biol. 17, 398–406. Forrester, W.C., Thompson, C., Elder, J.T., Groudine, M., 1986. A developmentally stable chromatin structure in the human b-globin gene cluster. Proc. Natl. Acad. Sci. USA 83, 1359–1363. Forrester, W.C., Tagegawa, S., Papayannopoulou, T., Stamatoyannnopoulos, T., Groudine, M., 1987. Molecular analysis of the human b-globin locus activation region. Proc. Natl. Acad. Sci. USA 86, 5439–5443. Fraser, P., Hurst, J., Collis, P., Grosveld, F., 1990. DNase hypersensitive sites 1, 2 and 3 of the human bglobin dominant control region direct position-independent expression. Nucl. Acid. Res. 18, 3503– 3508. Fraser, P., Pruzina, A.S., Antoniou, M., Grosveld, F., 1993. Each hypersensitive site of the human b-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev. 7, 106–113. Frier, J.A., Perutz, M.F., 1977. Structure of human foetal deoxyhaemoglobin. J. Mol. Biol. 12, 97–112. Fronticelli, C., Bucci, E., Orth, C., 1984. Solvent regulation of oxygen affinity in hemoglobin sensitivity of bovine hemoglobin to chloride ions. J. Biol. Chem. 259, 10841–10844. Gelin, B.R., Lee, A.W.M., Karplus, M., 1983. Hemoglobin tertiary structural change on ligand binding: its role in the cooperative mechanism. J. Mol. Biol. 171, 489–559. Giardina, B., Messana, I., Scatena, R., Castagnola, M., 1995. The multiple functions of hemoglobin. Crit. Rev. Biochem. Mol. Biol. 30, 165–196. Gibson, Q.H., Antonini, E., 1963. Rates of reaction of native human globins with some hemes. J. Biol. Chem. 238, 1384–1388. Go, M., 1981. Correlation of DNA exonic regions with protein structural units in hemoglobin. Nature 291, 90–92. Grosveld, F., Van Assendelfft, G.B., Greaves, D., Kollias, G., 1987. Position independent high level expression of the human b-globin gene in transgenic mice. Cell 51, 975–985. Grosveld, F., DeBoer, E., Dillon, F., 1989. The dynamics of globin gene expression and gene therapy results. Ann. N.Y. Acad. Sci. 850, 18–27. Grosveld, F., Antoniou, M., Berry, M., deBoer, E., Dillon, N., Ellis, J., Fraser, P., Hanscombe, O., Hurst, J., Imann, A., Lindenbaumm, M., Philipsen, S., Pruzina, S., Strouboulis, J., Raguz-Bolognesi, S., Talbot, D., 1993a. The regulation of human globin gene switching. Philos. Trans. R. Soc. London 339, 183–191. Grosveld, F., Dillon, N., Higgs, D., 1993b. The regulation of human gene expression. Baillieres Clin. Haematol. 6, 31–55. Gumucio, D.L., Shelton, D.A., Blanchard-McQuate, K., Gray, T.A., Tarle, S.A., Heilstedt-Williamson, H., Slighton, J., Collins, F.S., Goodman, M., 1994. Differential phylogenetic footprinting as a means to identify base changes responsible for recruitment of the anthropoid c gene to a fetal expression pattern. J. Biol. Chem. 269, 15371–15380. Guy, L.G., Mei, Q., Perkins, A.C., Orkin, S.H., 1998. Erythroid Kruppel-like factor is essential for beta globin gene expression even in the absence of gene competition, but is not sufficient to induce the switch from gamma-globin to beta-globin gene expression. Blood 91, 2259–2263. Handscombe, O., Whyatt, D., Fraser, P., Yannoutsos, N., Greaves, D., Dillon, N., Grosveld, F., 1991. Importance of globin gene order for correct developmental expression. Genes Dev. 5, 1387–1394. Hardison, R., 1998. Hemoglobins from bacteria to man: evaluation of different patterns of gene expression. J. Exp. Biol. 201, 1099–1117. Hardison, R., Slighton, J.L., Gumucio, D.L., Goodman, M., Stojanovic, N., Miller, W., 1997. Locus control regions of mammalian b-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insight. Gene 205, 73–94. Hargrove, M.S., Olson, J.S., 1996. The stability of holomyoglobin is determined by heme affinity. Biochem. 35, 11310–11318. Hashimoto, M., Ishimori, K., Imai, K., Miyuzaki, G., Moritomo, H., Wada, Y., Morishima, I., 1993. Site directed mutagenesis in hemoglobin: functional and structural studies of the intersubunit hydrogen

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

335

bond of threonine )38(C3) alpha-1–beta-1 interface in human hemoglobin. Biochemistry 32, 13688– 13695. He, Z., Russell, J.E., 2001. Expression, purification and characterization of human hemoglobin’s Gower 1 ðf2 e2 Þ, Gower 2 ða2 e2 Þ and Portland 2 ðf2 b2 Þ assembled in complex transgenic-knockout mice. Blood 97, 1099–1105. He, Z., Lian, L., Asakura, T., Russell, J.E., 2000. Functional effects of replacing human a and b globins with their embryonic globin homologues in defined haemoglobin heterotetramers. Br. J. Haematol. 109, 882–890. Hellegers, A.E., Schruefer, J.S., 1961. Nomograms and empirical equations relating oxygen tension, percentage saturation and pH in maternal and fetal blood. Am. J. Obstet. Gynecol. 81, 377–384. Henry, E.R., Jones, C.M., Hofrichter, J., Eaton, W.A., 1997. Can a two state MWC allosteric model explain hemoglobin kinetics? Biochemistry 36, 6511–6528. Hetch, F., Motulsky, A.G., Lemire, R.J., Shepard, T.E., 1966. Predominance of Hb Gower I in early embryonic development. Science 152, 91–92. Higgs, D.R., Wood, W.G., Jarman, A.P., Sharpe, J., Lida, J., Pretorius, I.M., Ayyub, H., 1990. A major positive regulatory region is located far upstream of the human a-globin gene cluster. Genes Dev. 4, 1588–1601. Hill, A.V., 1910. The possible effects of aggregation of the molecules of haemoglobin on its oxygen dissociation curve. J. Physiol. 40, IV–VII. Hill, E.P., Hill, J.R., Power, G.G., Longo, L.D., 1977. Carbon monoxide exchange between the human fetus and mother: a mathematical model. Amer. J. Physiol. 232, H311–H323. Ho, C., Russu, I.M., 1987. How much do we know about the Bohr effect of hemoglobin? Biochemistry 26, 6229–6305. Ho, P.J., 1999. The regulation of b-globin gene expression and b thalassaemia. Pathology 31, 315– 324. Hofmann, O.M., Brittain, T., 1996. Ligand binding kinetics and dissociation of the human embryonic haemoglobins. Biochem. J. 315, 65–70. Hofmann, O.M., Brittain, T., 1998. Partitioning of oxygen and carbon monoxide in the three human embryonic hemoglobins. Hemoglobin 22, 313–319. Hofmann, O.M., Mould, R., Brittain, T., 1995a. Allosteric modulation of oxygen binding to the three human embryonic haemoglobins. Biochem. J. 306, 367–370. Hofmann, O.M., Carrucan, G., Robson, N., Brittain, T., 1995b. The chloride effect in human embryonic haemoglobins. Biochem. J. 309, 959–962. Hofmann, O.M., Brittain, T., Wells, R.M.G., 1997. The control of oxygen affinity in the three human embryonic haemoglobins by respiration linked metabolites. Biochem. Mol. Biol. Int. 42, 553–566. Hopfield, J.J., Shulman, R.G., Ogawa, S., 1971. An allosteric model of hemoglobin: I Kinetics. J. Mol. Biol. 61, 425–443. Huang, B.L., Fan-Chiang, I.R., Wen, S.C., Koo, W.Y., Gavva, N.R., Shen, C.K.J., 1998. Derepression of human embryonic f globin promoter by a locus control region sequence. Proc. Natl. Acad. Sci. USA 95, 14669–14674. Huehns, E.R., 1974. The structure and function of haemoglobin: clinical disorders due to abnormal haemoglobin structure. In: Hardiston, R.M., Weatherall, D.J. (Eds.), Blood and its Disorders. Blackwell Scientific, Oxford, UK. Huehns, E.R., Farouqui, A.M., 1975. Oxygen dissociation properties of human embryonic red cells. Nature 254, 335–337. Huisman, T.H.J., Schroeder, W.A., Efremov, G.D., 1974. The present status of the heterogeneity of fetal hemoglobin in b thalassaemia: an attempt to unify some observations in thalassaemia and related conditions. Ann. N.Y. Acad. Sci. 232, 107–124. Huisman, T.H.J., Harris, H., Gravely, M., Schroeder, W.A., Shelton, J.R., Shelton, J.B., Evans, L., 1977. The chemical heterogeneity of fetal hemoglobin in normal newborn infants and in adults. Mol. Cell. Biol. 17, 45–55. Hultquist, D.E., Dean, R.T., Douglas, R.H., 1974. Homogeneous cytochrome b5 from human erythrocytes. Biochem. Biophys. Res. Commun. 60, 28–34.

336

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

Igarashi, K., Kataoka, K., Itoh, K., Hayashi, N., Nishizawa, M., Yamamoto, M., 1994. Regulation of transcription by dimerisation of erythroid factor NF-E2 with small Maf proteins. Nature 367, 568–572. Ikonomni, P., Noguchi, C.T., Miller, W., Kassahun, H., Hardison, R., Schechter, A.N., 2000. Levels of GATA-1/GATA-2 transcription factors modulate expression of embryonic and fetal hemoglobins. Gene 261, 277–287. Imai, K., 1982. In: Allosteric Effects in Haemoglobin. Cambridge University Press, Cambridge, UK, p. 33. Jackson, J.D., Miller, W., Hardison, R.D., 1996a. Sequences within and flanking hypersensitive sites 3 and 2 of the b-globin locus control region are required for synergistic versus additive interaction with the e globin gene reporter. Nucl. Acid. Res. 24, 4327–4335. Jackson, J.D., Petrykowska, H., Philipsen, S., Miller, W., Hardison, R., 1996b. Role of DNA sequences outside the cores of DNase hypersensitive sites (HS’s) in functions of the b-globin locus control region: domain opening and synergism between HS2 and HS3. J. Biol. Chem. 271, 11871–11878. Jaffe, E.R., 1964. In: Bishop, C., Surgenor, D.M. (Eds.), The Red Blood Cell. Academic Press, New York, USA, pp. 397–422. Jagus, R., Anderson, W.F., Safer, B., 1981. The regulation of initiation of mammalian protein synthesis. Prog. Nucl. Acid. Res. 25, 127–185. Jane, S.M., Cunningham, J.M., 1996. Molecular mechanisms of hemoglobin switching. Int. J. Biochem. Cell. Biol. 28, 1197–1209. Jane, S.M., Cunningham, J.M., 1998. Understanding fetal globin gene expression: a step towards effective HbF reactivation in haemoglobinopathies. Br. J. Haematol. 102, 415–422. Jane, S.M., Ney, P.A., Vanin, E.F., Gumucio, D.L., Nienhuis, A.W., 1992. Identification of a stage selector element in the human gamma globin gene promoter that fosters preferential interactions with the 5’ HS2 enhancer when in competition with the beta promoter. EMBO J. 11, 2961–2969. Janin, J., Wodak, S.J., 1993. The quaternary structure of carbonmonoxy hemoglobin Ypsilanti. Proteins 15, 1–4. Jarman, A.P., Woo, W.G., Sharpe, J.A., Gourdon, G., Ayyub, H., Higgs, D.R., 1991. Characterisation of the major regulatory element upstream of the human alpha globin gene cluster. Mol. Cell. Biol. 11, 4679–4689. Jensen, M., Attenberger, H., Schneider, C.H., Walther, J.U., 1982. The developmental changes in the G c and A c globin proportions in hemoglobin F. Eur. J. Pediatr. 138, 311–314. Jue, T., LaMar, G.N., 1984. Heme orientational heterogeneity in deuterohemin reconstituted horse and human hemoglobin characterised by proton NMR spectroscopy. Biochem. Biophys. Res. Commun. 119, 5203–5206. Kachalova, G.S., Popov, A.N., Bartunik, H.D., 1999. A steric mechanism for inhibition of CO binding to heme proteins. Science 284, 473–476. Kadonaga, J.T., 1998. Eukaryotic transcription: an interlaced network of transcription factors and chromatin-modifying machines. Cell 92, 307–313. Katz, D.S., White, S.P., Huang, W., Kumar, R., Christianson, D.W., 1994. Structure determination of aquomet porcine hemoglobin at 2.8 A resolution. J. Mol. Biol. 224, 541–553. Kellet, G.L., Gutfreund, H., 1970. Reactions of haemoglobin dimers after ligand dissociation. Nature 227, 921–926. Kidd, R.D., Mathews, A., Baker, H.M., Brittain, T., Baker, E.N., 2001a. Subunit dissociation and reassociation leads to preferential crystallization of haemoglobin Barts ðc4 Þ from solutions of human embryonic haemoglobin Portland ðf2 c2 Þ at low pH. Acta Cryst. D57, 921–924. Kidd, R.D., Baker, H.M., Mathews, A., Brittain, T., Baker, E.N., 2001b. Oligomerisation and ligand binding in a homotetrameric hemoglobin: Two high-resolution crystal structures of hemoglobin Barts ðc4 Þ a marker for a-thalassaemia. Prot. Sci. 10, 1739–1749. Kidd, R.D., Russell, J.E., Watmough, N.J., Baker, E.N., Brittain, T., 2001c. The role of b chains in the control of hemoglobin oxygen binding function: chimeric human/mouse proteins, structure and function. Biochemistry 40, 15669–15675. Kidd, R.D., Baker, E.N., Brittain, T., 2002. A modeling study of the interaction and electron transfer between cytochrome b5 and some oxidized haemoglobins. J. Biol. Inorg. Chem. 7, 23–30.

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

337

Kilmartin, J.V., 1974. Influence of DPG on the Bohr effect of human haemoglobin. FEBS. Lett. 38, 147– 148. Kilmartin, J.V., 1977. The Bohr effect of human hemoglobin. TIBS (November), 247–250. Kilmartin, J.V., Rossi-Bernardi, L., 1969. Inhibition of CO2 combination and reduction of the Bohr effect in haemoglobin chemically modified at its a amino groups. Nature 222, 1243–1246. Kilmartin, J.V., Rossi-Bernardi, L., 1973. Interaction of hemoglobin with hydrogen ions, carbon dioxide and organic phosphates. Physiol. Rev. 53, 836–890. Kilmartin, J.V., Breen, J.J., Roberts, G.C.K., Ho, C., 1973. Direct measurement of the pK values of an alkaline Bohr effect group in human haemoglobin. Proc. Natl. Acad. Sci. USA 70, 1246–1249. Kilmartin, J.V., Imai, K., Jones, R.T., Farouqui, A.R., Fogg, J., Baldwin, JM., 1978. Role of Bohr group salt bridges in cooperativity in hemoglobin. Biochim. Biophys. Acta 534, 15–25. Komar, A.A., Kommer, A., Krasheninnikov, I.A., Spirin, A.S., 1993. Cotranslational heme binding to nascent globin chains. FEBS. Letts. 326, 261–263. Koshland, D.E., Nemethy, G., Filmer, D., 1966. Comparison of experimental binding data and theoretical models in protein containing subunits. Biochemistry 5, 365–385. Kuzolik, A.E., Sail, S., Bellam-Koch, A., Bartram, C.R., Kohn, E., Kleihauer, E., 1991. The proximal element of the b-globin locus control region is not functionally required in vivo. J. Clin. Invest. 87, 2142–2146. LaMar, G.N., Yamamoto, Y., Jue, T., Smith, K.M., Pandey, R.K., 1985. H1 NMR characterization of metastable and equilibrium heme orientation heterogeneity in reconstituted and native hemoglobin. Biochemistry 24, 3826–3831. Lavalette, D., Tetreau, C., Mispelter, J., Momenteau, M., Lhoste, J.M., 1984. Linear free energy relationships in binding of oxygen and carbon monoxide with heme model compounds and heme proteins. Eur. J. Biochem. 145, 555–566. Lee, W., Haslinger, A., Karin, M., Tjian, R., 1987. Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40. Nature 325, 368– 372. Lewis, S.M., 1974. The constituents of normal blood. In: Hardiston, R.M., Weatherall, D.J. (Eds.), Blood and its Disorders. Blackwell Scientific, Oxford, UK, pp. 3–68. Ley, T.J., Desimone, J., Anagou, N.P., Keller, G.H., Humphries, K., Turner, P.H., Young, N.S., Heller, P., Nienhuis, A.W., 1982. 5-azacytidine selectively increases gamma globin synthesis in a patient with beta thalassaemia. New. Eng. J. Med. 307, 1469–1475. Ley, T.J., DeSimone, J., Anagnou, N.P., Keller, G.H., Humphries, R.K., Turner, P.H., Young, N.S., Heller, P., Niehuis, A.W., 1983. 5-azacytidine increases c globin synthesis and reduces the proportion of dense cells in patients with sickle cell anaemia. Blood 62, 370–380. Li, J., Noguchi, C.T., Miller, W., Hardison, R., Schechter, A.N., 1998. Multiple regulatory elements in the 50 -flanking sequence of the human epsilon globin gene. J. Biol. Chem. 273, 10202–10209. Li, Q., Blau, C.A., Clegg, C.H., Rohde, A., Stamatoyannopoulos, G., 1989. Multiple epsilon-promoter elements participate in the developmental control of epsilon-globin genes in transgenic mice. J. Biol. Chem. 273, 17361–17367. Li, Q., Stamatoyannopoulos, G., 1994. Hypersensitive site 5 of the human b globin locus control region functions as a chromatin insulator. Blood 84, 1399–1401. Liddington, R.C., Derewenda, Z., Dodson, G., Harris, D., 1988. Structure of liganded T-state of hemoglobin identifies the origin of co-operative oxygen binding. Nature 331, 725–728. Lim, M., Jackson, T.A., Anfinrud, P.A., 1997. Modulating carbon monoxide binding affinity and kinetics in myoglobin: the role of the distal histidine and heme pocket docking site. J. Biol. Inorg. Chem. 2, 531–536. Longo, L.D., 1970. Carbon monoxide in the pregnant mother and fetus and its exchange across the placenta. Ann. N.Y. Acad. Sci. 174, 313–341. Lopez, R.A., Schoetz, S., DeAngelis, K., O’Neill, D., Bank, A., 2002. Multiple hematopoietic defects and delayed globin switching in Ikaros null mice. Proc. Natl. Acad. Sci. USA 99, 602–607. Lowenhaupt, K., Linger, J.B., 1978. A change in the stability of globin mRNA during the induction of murine erythroleukemic cells. Cell 14, 337–344.

338

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

Lukin, J.A., Simplaceanu, V., Zou, M., Ho, N.T., Ho, C., 2000. NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin. Proc. Natl. Acad. Sci. USA 97, 10354–10358. Manning, J.M., Dumoulin, A., Manning, L.R., Chen, W., Padovan, J.C., Chait, B.T., Popowicz, A., 1999. Remote contributions to subunit interactions: lessons from adult and fetal hemoglobin. TIBS 24, 211–212. Mansouri, A., Winterhalter, K., 1973. Non equivalence of chains in hemoglobin oxidation. Biochemistry 12, 4946–4949. Martin, D.K., Zon, L.I., Mutter, G., Orkin, S.H., 1990. Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages. Nature 344, 444–447. Mathews, A.J., Brittain, T., 2001. Haem rotational disorder in recombinant and reticulocyte derived haemoglobins: evidence for a directed haem insertion reaction in eukaryotes. Biochem. J. 357, 305–311. Mellon, P., Parker, V., Gluzman, Y., Maniatis, T., 1981. Identification of DNA sequences required for transcription of human alpha-globin gene in a new SV40 host–vector system. Cell 27, 279–288. Mignotte, V., Wall, L., deBoer, E., Grosveld, F., Romeo, P.H., 1989. Two tissue specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene. Nucl. Acid. Res. 17, 37–54. Miller, I.J., Bieker, J.J., 1993. A novel erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol. Cell. Biol. 13, 2776–2786. Milot, E., Strouboulis, J., Trimborn, T., Wijgerde, M., deBoer, E., Langveld, A., Tan-Un, K., Vergeer, W., Yannoutos, N., Grosveld, F., Fraser, P., 1996. Heterochromatin effects on the frequency and duration of LCR mediated gene transcription. Cell 87, 105–114. Moi, P., Chan, K., Asunis, I., Cao, A., Kan, Y.W., 1994. Isolation of NF-E2 related factor 2 (Nrf2) a NFE2 like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the b-globin locus control region. Proc. Natl. Acad. Sci. USA 91, 9926–9930. Moldave, K., 1985. Eukaryotic protein synthesis. Ann. Rev. Biochem. 54, 1109–1151. Monod, J., Wyman, J., Changeaux, J.P., 1965. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118. Morley, B.J., Abbot, C.A., Sharpe, J.A., Lida, J., Chan-Thomas, P.S., Wood, W.G., 1992. A single bglobin locus control region element (50 hypersensitive site 2) is sufficient for developmental regulation of human globin genes in transgenic mice. Mol. Cell. Biol. 12, 2057–2066. Nagel, R.L., Gibson, Q.H., 1971. The binding of hemoglobin to haptoglobin and its relation to subunit dissociation of hemoglobin. J. Biol. Chem. 246, 69–73. Ney, P.A., Sorrentino, B.P., Donagh, K.T., Nienhuis, A.W., 1990. Tandem AP-1-binding sites within the human b-globin dominant control region function as an inducible enhancer in erythroid cells. Genes Dev. 4, 993–1006. Nienhuis, A.W., Benz, E.J., 1977. Regulation of hemoglobin synthesis during the development of the red cell. New. Engl. J. Med. 297, 1318–1328. Nienhuis, A.W., Maniatis, T., 1987. Structure and expression of globin genes in erythroid cells. In: Stamatoyannopoulos, G., Nienhuis, A.W., Leder, P., Majeurs, P.W. (Eds.), The Molecular Basis of Blood Diseases. W.B. Saunders, Philadelphia, USA, pp. 28–66. Nigen, A.M., Manning, J.M., Alben, J.O., 1980. Oxygen linked binding sites for inorganic ions. J. Biol. Chem. 255, 5525–5529. Northrup, S.H., Boles, J.O., Reynolds, J.C.L., 1987. Electrostatic effects in the Brownian dynamics of association and orientation of heme proteins. J. Phys. Chem. 91, 5991–5998. Northrup, S.H., Boles, J.O., Reynolds, J.C.L., 1988. Brownian dynamics of cytochrome c and cytochrome c peroxidase association. Science 241, 67–70. O’Donnel, S., Mandaro, R., Schuster, T., Arnone, A., 1979. X ray diffraction and solution studies of specifically carbamylated human hemoglobin A. J. Biol. Chem. 254, 12204–12208. Olson, J.S., Mathews, A.J., Rohlfs, R.J., Springer, B.A., Egeberg, K.D., Sligar, S.G., Tame, J., Renaud, J.P., Nagai, K., 1988. The role of the distal histidine in myoglobin and hemoglobin. Nature 336, 265– 266. Orkin, S.H., 1990. Globin gene regulation and switching. Cell 63, 665–672. Orkin, S.H., 1995. Regulation of globin gene expression in erythroid cells. Eur. J. Biochem. 231, 271–281. Passon, G.P., Hultquist, D.E., 1972. Soluble cytochrome b5 reductase from erythrocytes. Biochim. Biophys. Acta 275, 62–73.

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

339

Pataryas, H.A., Stamatoyannopoulos, G., 1972. Haemoglobin in human fetuses: evidence for adult haemoglobin production after the 11th gestation week. Blood 39, 688–696. Perez-Stables, C., 1994. Distinct negative mechanisms involved in the repression of human embryonic e and fetal G c globin genes in transgenic mice. J. Biol. Chem. 269, 33109–33115. Perkins, A.C., Sharpe, A.H., Orkin, S.H., 1995. Lethal b-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature 375, 318–322. Perkins, A.C., Gaensler, K.M.L., Orkin, S.H., 1996. Silencing of human fetal globin expression is impaired in the absence of the adult b-globin gene activator protein EKLF. Proc. Natl. Acad. Sci. USA 93, 12267–12271. Perrine, S.P., Rudolph, A., Faller, D.V., Roman, C., Cohen, R.A., 1988. Butyrate infusions in the ovine fetus delay the biological clock for globin gene switching. Proc. Natl. Acad. Sci. USA 85, 8540– 8542. Perrine, S.P., Miller, B.A., Faller, D.V., Cohen, R.A., Vichinsky, E.P., Hurst, D., Lubin, B.H., Papayannopoulou, 1989. Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with HbSS and b thalassaemia. Blood 74, 454–459. Perutz, M.F., 1970. Steriochemistry of co-operative effects in hemoglobin. Nature 228, 726–739. Perutz, M.F., 1972. Haem–haem interactions. Nature 237, 495–499. Perutz, M.F., 1979. Regulation of oxygen affinity of haemoglobin: influence of structure of the globin on the haem. Ann. Rev. Biochem. 48, 327–386. Perutz, M.F., 1989. Mechanisms of Co-operativity and Allosteric Regulation in Proteins. Cambridge University Press, Cambridge, UK. Perutz, M.F., Rossman, M.G., Cullis, A.F., Muirhead, H., Will, G., North, A.C.T., 1960. Structure of haemoglobin. A three-dimensional Fourier structure at 2 A resolution. Nature 185, 416–422. Perutz, M.F., Muirhead, H., Mozzarelli, L., Crowther, R.A., Greer, J., Kilmartin, J.V., 1969. Identification of residues responsible for the alkaline Bohr effect in haemoglobin. Nature 222, 1240–1243. Perutz, M.F., Kilmartin, J.V., Nishikura, K., Fogg, J.H., Butler, P.J.G., Rollema, H.M.S., 1980. Identification of residues contributing to the Bohr effect in human haemoglobin. J. Mol. Biol. 138, 649–672. Perutz, M.F., Fermi, G., Luisi, B., Shaana, B., Liddington, R.C., 1987. Stereochemistry of co-operative effects in hemoglobin. Accs. Chem. Res. 20, 309–321. Perutz, M.F., Fermi, G., Poyart, C., Pagnier, J., Kister, J., 1993. A novel allosteric mechanism in haemoglobin: structure of bovine deoxyhaemoglobin, absence of specific chloride binding sites and origin of chloride linked Bohr effect in bovine and human haemoglobin. J. Mol. Biol., 233,536–545. Perutz, M.F., Shih, D.T.B., Williamson, D., 1994. The chloride effect in human haemoglobin. A new kind of allosteric mechanism. J. Mol. Biol. 239, 555–560. Peters, B., Merezhinskaya, N., Diffley, J.F., Noguchi, C.T., 1993. Protein-DNA interactions in the eglobin gene silencer. J. Biol. Chem. 268, 3430–3437. Petersen, K., Clegg, C., Navas, P., Norton, E., Kimbrough, T., Stamatoyannopoulos, G., 1996. Effect of deletion of 50 HS3 or 50 HS2 of the human b-globin LCR on the developmental regulation of globin gene expression in b-YAC transgenic mice. Proc. Natl. Acad. Sci. USA 93, 6605–6609. Peterson, C.L., Tamkun, J.W., 1995. The SW1–SNF complex: a chromatin remodeling machine? TIBS 20, 143–146. Peterson, K.R., Stamatoyannopoulos, G., 1993. Role of gene order in developmental control of human gamma-and beta-globin gene expression. Mol. Cell. Biol. 13, 4836–4843. Philipsen, S., Pruzina, S., Grosveld, F., 1993. The minimal requirements for activity in transgenic mice of hypersenstive site 3 of the b-globin locus control region. EMBO J. 12, 1077–1085. Poncz, M.P., Heuthorn, P., Stockert, C., Surrey, S., 1989. Globin gene expression in hereditary persistence of fetal hemoglobin and db°-thalassaemia. In: McLean, N. (Ed.), Oxford Surveys of Eukaryotic Genes. Oxford University Press, Oxford, pp. 163–203. Pondel, M.D., Proudfoot, N.J., Whitelaw, C., Whitelaw, E., 1992. The developmental regulation of the human zeta globin gene in transgenic mice employing beta-galactosidase as a reporter gene. Nucl. Acid. Res. 20, 5655–5660. Raich, N., Papayannopolou, T., Stamatoyannopoulos, G., Enver, T., 1992. Demonstration of a human e globin gene silencer with studies in transgenic mice. Blood 79, 861–864.

340

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

Raich, N., Clegg, C.H., Grofti, J., Romeo, P.H., Stamatoyannopoulos, G., 1995. GATA-1 and YY1 are developmental repressors of the human e-globin gene. EMBO J. 14, 801–809. Robertson, G., Garrick, D., Wu, W., Kearns, M., Martin, D., Whitlam, E., 1995. Position dependent variegation of globin transgene expression in mice. Proc. Natl. Acad. Sci. USA 92, 5371–5375. Robson, N., Brittain, T., 1996a. Conformational relaxation and ligand binding to singly reduced human embryonic hemoglobins. Biochem. Mol. Biol. Int. 38, 1245–1253. Robson, N., Brittain, T., 1996b. Heme stability in human embryonic haemoglobins. J. Inorg. Biochem. 64, 137–147. Rose, M.Y., Olson, J.S., 1983. The kinetic mechanism of heme binding to human apo-hemoglobin. J. Biol. Chem. 258, 4298–4303. Ross, J., 1995. mRNA stability in mammalian cells. Microbiol Rev. 59, 423–450. Rossi-Fanelli, A., Antonini, E., 1958. Studies on the oxygen and carbon monoxide equilibria of human myoglobin. Arch. Biochem. Biophys. 77, 478–492. Royer, W.E., Knapp, J.R.E., Strand, K., Heaslet, H.A., 2001. Co-operative hemoglobins: conserved fold, diverse quaternary assemblies and allosteric mechanisms. TIBS 26, 297–304. Russell, J.E., Liebhaber, S.A., 1998. Reversal of lethal a and b thalassaemias in mice by expression of human embryonic globins. Blood 92, 3057–3063. Russu, I.M., Ho, N.T., Ho, C., 1982. A proton NMR investigation of histidyl residues in human normal adult hemoglobin. Biochemistry 21, 5031–5043. Ryan, T.M., Behringer, R.R., Martin, N.C., Townes, T.M., Palmiter, R.D., Brinster, R.C., 1989. A single erythroid specific DNase I super-hypersensitive site activates high levels of human b-globin gene expression in transgenic mice. Genes Dev. 3, 314–323. Sabath, D., Koehler, K.M., Yang, W.Q., Stamatoyannopoulos, G., 1995. Identification of a major positive regulatory element located 50 to the human zeta globin gene. Blood 85, 2587–2597. Sachs, A.B., 1993. Messenger RNA degradation in eukaryotes. Cell 74, 413–421. Safer, B., 1983. Regulation of the catalytic utilization of eIF-2. Cell 33, 7–8. Safer, B., Jagus, R., Konieczny, A., Crouch, D., 1982. The mechanism of translational inhibition in hemindeficient lysates. In: Grunberg-Manago, M., Safer, B. (Eds.), Interaction of Translational and Transcriptional Controls in the Regulation of Gene Expression. Elsevier, New York. Scheepens, A., Mould, R., Hofmann, O., Brittain, T., 1994. Some effects of post-translational N-terminal acetylation of human embryonic f globin protein. Biochem. J. 310, 597–600. Schroeder, W.A., 1980. The synthesis and chemical heterogeneity of human fetal hemoglobin. Hemoglobin 4, 431. Schroeder, W.A., Huisman, T.H.J., Brown, A.K., Uy, R., Bouver, N.G., Lerch, P.O., Shelton, J.R., Shelton, J.B., Apell, G., 1971. Postnatal changes in chemical heterogeneity of human fetal hemoglobin. Pediatr. Res. 5, 493–499. Schumaker, M.A., Zhelenznova, E.E., Poundstone, K.S., Kluger, R., Jones, R.T., Brennan, R.G., 1997. Allosteric intermediates indicate R2 is the liganded hemoglobin end state. Proc. Natl. Acad. Sci. USA 94, 7841–7844. Shaanan, B., 1983. Structure of human oxyhaemoglobin at 2.1 A resolution. J. Mol. Biol. 171, 31–59. Sharpe, J.A., Chan-Thomas, P.S., Lida, J., Ayyub, H., Wood, W.G., Higgs, D.R., 1992. Analysis of the human a-globin upstream regulatory element (HS-40) in transgenic mice. EMBO J. 11, 4565– 4572. Sharpe, J.A., Wells, D.J., Whitelaw, E., Vyas, P., Higgs, D.R., Wood, W.G., 1993. Analysis of the human a-globin gene cluster in transgenic mice. Proc. Natl. Acad. Sci. USA 90, 11262–11266. Shih, D.M., Wall, R.J., Shapiro, S.G., 1990. Developmentally regulated and erythroid specific expression of the human embryonic b-globin gene in transgenic mice. Nucl. Acid. Res. 18, 5465–5472. Shih, D.T.B., Luisi, B.F., Miyazaki, G., Perutz, M.F., Nagai, K., 1993. A mutagenic study of the allosteric linkage of His (HC 3) 146 b in haemoglobin. J. Mol. Biol. 230, 1291–1296. Sigfridsson, E., Ryde, U., 1999. On the significance of hydrogen bonds for the discrimination between carbon monoxide and oxygen by myoglobin. J. Biol. Inorg. Chem. 4, 99–110. Silva, M.M., Rogers, P.H., Arnone, A., 1992. A third quaternary structure of human hemoglobin A at 1.7 A resolution. J. Biol. Chem. 267, 17248–17256.

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

341

Slebodnick, C., Ibers, J.A., 1997. Myoglobin models and steric origins of the discrimination between oxygen and carbon monoxide. J. Biol. Inorg. Chem. 2, 521–525. Smith, Z.E., Higgs, D.R., 1999. The pattern of replication at a human telometric region (16p 13.3): its relationship to chromosome structure and gene expression. Hum. Mol. Genet. 8, 1373–1386. Smith, F.R., Simmons, K.C., 1994. Cyanomet human hemoglobin crystallized under physiological conditions exhibits the Y quaternary structure. Prot. Struct. Funct. Genet. 18, 295–300. Smith, F.R., Latterman, E.E., Carter, C.W., 1991. The mutation b 99 Asp–Tyr stabilizes Y – a new composite quaternary state of human hemoglobin. Proteins 10, 81–91. Sposi, N.M., Zon, L.I., Core, A., Valtieri, M., Testa, U., Gabbianelli, M., Mariani, G., Bottero, L., Mather, C., Orkin, S.H., Peschle, C., 1992. Cycle dependent initiation and lineage dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors. Proc. Natl. Acad. Sci. USA 89, 6353–6357. Springer, B.A., Egeberg, K.D., Sligar, S.G., Rohlfs, R.J., Mathews, A.J., Olson, J.S., 1989. Discrimination between oxygen and carbon monoxide and inhibition of autoxidation by myoglobin. J. Biol. Chem. 264, 3057–3060. Srivanasan, R., Rose, G.D., 1994. The T–R transformation in hemoglobin: a reevaluation. Proc. Natl. Acad. Sci. USA 91, 11113–11117. Stamatoyannopoulos, G., Nienhuis, A.W., 1987. Hemoglobin switching. In: Stamatoyannopoulos, G., Nienhuis, A.W., Leder, P., Majeurs, P.W. (Eds.), The Molecular Basis of Blood Diseases. W.B. Saunders, Philadelphia, USA, pp. 66–105. Stegink, L.D., Meyer, P.D., Brummel, M.C., 1971. Human fetal hemoglobin F: acetylation status. J. Biol. Chem. 246, 3001–3007. Strauss, E.C., Andrews, N.C., Higgs, D.R., Orkin, S.H., 1992. In vivo protein DNA interactions at hypersensitive site 3 of the human beta globin locus control region. Mol. Cell. Biol. 12, 2135–2142. Sutherland-Smith, A.J., Baker, H.M., Hofmann, O.M., Brittain, T., Baker, E.N., 1998. Crystal structure of a human embryonic haemoglobin: the carbonmonoxy form of Gower II ða2 e2 Þ haemoglobin at 2.9 A resolution. J. Mol. Biol. 280, 475–484. Szabo, A., Karplus, M., 1972. A mathematical model for structure function relations in hemoglobin. J. Mol. Biol. 72, 163–197. Talbot, T., Collis, P., Antoniou, M., Vidal, M., Grosveld, F., Greaves, D.R., 1989. A dominant control region from the human b-globin locus conferring integration site-independent gene expression. Nature 338, 352–355. Talbot, D., Philipsen, S., Fraser, R., Grosveld, F., 1990. Detailed analysis of the site 3 region of the human b-globin dominant control region. EMBO J. 9, 2169–2178. Tame, J.R.H., 1999. What is the true structure of liganded haemoglobin? TIBS 24, 372–377. Terwari, R., Gillemans, N., Wijerde, M., Neuz, B., Lindern, M., Grosveld, F., Philipsen, S., 1998. Erythroid Kruppel-like factor is active in primitive and definitive erythroid cells and is required for the function of 50 HS3 of the beta globin locus control region. EMBO J. 17, 2334–2341. Townes, T., Behringer, R., 1990. Human globin locus activation region (LAR): role in temporal control. Trends Genet. 6, 219–223. Traylor, T.G., Berzinis, A.P., 1980. Binding of oxygen and carbon monoxide to hemes and hemoproteins. Proc. Natl. Acad. Sci. USA 77, 3171–3175. Traylor, T.G., Koga, N., Deardurff, L.A., Swepston, P.N., Ibers, J.A., 1984. 1,3 Adamantane-2 13porphyrin-6-6-cyclophane crystal structure of free base and steric effects on ligation of the iron-II complex. J. Am. Chem. Soc. 106, 5132–5143. Traylor, T.G., Tsuchiya, S., Campbell, D., Mitchell, M., Stynes, D., Koga, N., 1985. Anthracene heme cyclophanes steric effects in carbon monoxide, oxygen and isocyanide binding. J. Am. Chem. Soc. 107, 604–614. Trudel, M., Contantini, F., 1987. A 30 enhancer contributes to the stage-specific expression of the human b-globin gene. Genes Dev. 1, 954–961. Tsai, S.F., Martin, D.I.K., Zon, L.I., D’Andrea, A.D., Wong, G.G., Orkin, S.H., 1989. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature 339, 446–451.

342

T. Brittain / Molecular Aspects of Medicine 23 (2002) 293–342

Tuan, D., Solomon, W., London, I., Lee, D., 1989. An erythroid-specific developmental-stageindependent enhancer far upstream of the human ‘b-like globin’ genes. Proc. Natl. Acad. Sci. USA 86, 2554–2558. Tuchinda, S., Nagai, K., Lehman, H., 1975. Oxygen dissociation curve of haemoglobin Portland. FEBS. Lett. 49, 390–391. Van Beek, G.G.M., deBruin, S.H., 1980. Identification of the residues involved in the oxygen linked chloride ion binding sites in human deoxyhemoglobin and oxyhemoglobin. Eur. J. Biochem. 105, 353– 360. Van Heel, M., 1991. A new family of powerful multivariate statistical sequence analysis techniques. J. Mol. Biol. 220, 877–887. Volloch, V., Housman, D., 1981. Stability of globin mRNA in terminally differentiating mouse erythroleukemic cells. Cell 23, 509–514. Vyas, P., Vickers, M.A., Simmons, D.L., Ayyub, H., Craddock, C.F., Higgs, D.R., 1992. Cis-acting sequences regulating expression of the human a-globin cluster lie within constitutively open chromatin. Cell 69, 781–793. Wagenbach, M., O’Rourke, K., Vitez, L., Wieczorek, A., Hoffman, S., Durfee, S., Tedesco, J., Stetler, G., 1991. Synthesis of wild type and mutant human hemoglobin in Saccharomyces cerevisiae. BioTechnology 9, 57–61. Wallace, W.J., Maxwell, J.C., Caughey, W.S., 1974. Mechanism for the autoxidation of hemoglobin by phenol, nitrite and oxidant drugs: peroxide formation by one electron donation to bound oxygen. FEBS Lett. 43, 33–36. Wallace, W.J., Houtchens, R.A., Maxwell, J.C., Caughey, W.S., 1982. Mechanism of autoxidation of hemoglobins and myoglobins. J. Biol. Chem. 257, 4966–4977. Wang, X., Kiledjian, M., Weiss, I.M., Liebhaber, S., 1995. Detection and characterization of a 30 untranslated region ribonucleoprotein complex associated with human b-globin mRNA stability. Mol. Cell. Biol. 15, 1769–1777. Weiss, J.J., 1964. Nature of the iron–oxygen bond in oxyhemoglobin. Nature 202, 83–84. Weiss, I.M., Liebhaber, S.A., 1994. Erythroid cell-specific determinants of b-globin mRNA stability. Mol. Cell. Biol. 14, 8123–8132. Wiejgerde, M., Grosveld, F., Fraser, P., 1995. Transcription complex stability and chromatin dynamics in vivo. Nature 377, 209–213. Wintrobe, M.M., 1981. Clinical Hematology. Lea and Febiger, Philadelphia, USA. Wyman, J., 1948. Heme proteins. Adv. Prot. Chem. 4, 407–531. Wyman, J., 1964. Linked functions and reciprocal effects in hemoglobin: a second look. Adv. Prot. Chem. 19, 223–286. Wyman, J., 1967. Allosteric linkage. J. Am. Chem. Soc. 89, 2202–2218. Wyman, J., 1984. Linkage graphs: a study in thermodynamics of macromolecules. Q. Rev. Biophys. 17, 453–488. Yamamoto, Y., LaMar, G.N., 1986. H1 NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A. Biochemistry 25, 5288–5297. Yu, Z., Bock, J., Slighton, J., Villeponteau, B., 1994. A 50 matrix-attachment region and the polyoma enhancer together confer position-independent transcription. Gene 139, 139–145. Zhang, Q., Reddy, P.M.S., Yu, C.Y., Bastiani, C., Higgs, D., Stamatoyannopoulos, G., Papayannopoulou, T., Shen, C.K.J., 1993. Transcriptional activation of human zeta-2 globin promoter by the alpha globin regulatory element (HS40): functional role of specific nuclear factor-DNA complexes. Mol. Cell. Biol. 13, 2298–2308. Zhang, Q., Rombel, I., Reddy, G.N., Shen, C.K.Y., 1995. In: Stamatoyannopoulos, G. (Ed.), Molecular Biology of Hemoglobin Switching. Intercept Ltd., Hampshire, UK, pp. 193–202. Zheng, T., Brittain, T., Watmough, N.J., Weber, R.E., 1999. The role of amino acid a38 in the control of oxygen binding to human adult and embryonic haemoglobin Portland. Biochem. J. 343, 681–685.