Biomedical subjects

D T Shih

Publications and source records attributed to D T Shih.

At least 19 recordsLinked to original sources

PubMed1998

Adenoviral gene transfer of beta3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma.

Expression of the beta3 subunit of the alphavbeta3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of alphavbeta3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing alphavbeta3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional alphavbeta3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, alphavbeta3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and-invasive vertical growth phase primary melanoma.

Adenoviridae↗

PubMed1998

Hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, codon 143 CAC-->TAC--a variant with altered oxygen affinity that compromises measurement of glycated hemoglobin in diabetes mellitus: structure, function, and DNA sequence.

OBJECTIVE: To determine the nature and characteristics of a unique hemoglobin variant that causes a spurious increase in glycated hemoglobin (HbA1c). MATERIAL AND METHODS: Blood specimens from four unrelated persons with this hemoglobin variant were examined by conventional laboratory methods, including electrophoresis, high-performance ion-exchange chromatography, and isoelectric focusing; by amino acid sequence analysis, polymerase chain reaction-based DNA sequence analysis, and electrospray ionization mass spectrometry, to establish the molecular structure; and by studies of oxygen affinity under varied conditions, to define the functional characteristics of the hemoglobin variant. RESULTS: The unique hemoglobin variant observed in these four cases is due to the mutation CAC-->TAC, at beta-globin gene codon 143, corresponding to beta 143 (H21) His-->Tyr. This amino acid substitution affects an important 2,3-diphosphoglycerate binding site and slightly increases the oxygen affinity of the hemoglobin variant. CONCLUSION: A hitherto unrecognized hemoglobin variant, encountered in four unrelated persons of Irish or Scots-Irish ancestry, hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, has now been characterized at the molecular, structural, and functional levels. Although it is associated with a slight increase in oxygen affinity, it is without hematologic effect, and its only clinical significance is that it coelutes with HbA1c on ion-exchange chromatography and thereby causes a spurious increase in HbA1c and compromises the use of this analyte to monitor the treatment of diabetes mellitus.

Adult↗

PubMed1997

EGF-R dependent regulation of keratinocyte survival.

Tissue organization and maintenance within multicellular organisms is in part dependent on the ability of cells to undergo programmed cell death or apoptosis. Conversely, disruption of cell death pathways often is associated with tumor development. At present, the molecular control of apoptosis in epithelial cells is poorly understood. Here we describe evidence linking epidermal growth factor-receptor (EGF-R) activation to survival of normal human keratinocytes in culture. Inhibition of EGF-R activation by an anti-EGF-R antagonistic monoclonal antibody (mAb 425), followed by detachment of keratinocytes from the substratum, induced extensive death with several features of apoptosis in keratinocyte cultures. Other, non-epithelial normal human cells including melanocytes and fibroblasts, did not show this effect. Similar to EGF-R blockade by mAb 425, inhibition of the EGF-R tyrosine kinase activity using tyrphostin AG1478 resulted in lack of attachment and extensive cell death upon passaging. Attachment to keratinocyte-derived ECM partially resuced mAb 425-treated keratinocytes from cell death, indicating that adhesion-dependent and EGF-R-dependent signal transduction pathways serve partially overlapping but not redundant roles in supporting keratinocyte survival.

Apoptosis↗

PubMed1997

Epitopes of adhesion-perturbing monoclonal antibodies map within a predicted alpha-helical domain of the integrin beta 1 subunit.

Several recent studies have demonstrated the involvement of various domains of the beta 1 integrin subunit in ligand binding. Thus, specific amino acids have been shown to be important in divalent cation binding, and others have been implicated by peptide crosslinking to play an intimate role in integrin-ligand interactions. Added to these data are previous observations that a group of adhesion-blocking anti-chicken beta 1 antibodies mapped within the first 160 amino acid residues of the subunit. These observations suggested that this region plays a critical role in integrin ligand recognition. In order to further define the domain in which the epitopes for these antibodies are clustered, a series of mouse/chicken chimeric beta 1 constructs were examined for their reactivity with each of these antibodies. Most of the antibodies recognize a region between residues 124 to 160 of the chicken beta 1 subunit. Computer modeling predicted a possible amphipathic alpha-helical configuration for the region between residues 141 to 160. Consistent with this prediction, circular dichroism and NMR analysis revealed a tendency for a synthetic peptide containing these residues to form an alpha-helix. The significance of this structural characteristic was demonstrated by a mutation at residue 149 that disrupted the alpha-helix formation and resulted in a loss of the ability to form heterodimers with alpha subunits, localize to focal contacts, or be transported to the cell surface. The direct involvement of residues 141 to 160 in ligand binding was supported by the ability of a peptide with this sequence to elute integrins from a fibronectin affinity column. Thus, our data suggest that residues 141 to 160 of the integrin beta 1 subunit, when arranged in an alpha-helix configuration, participate in ligand binding.

3T3 Cells↗

PubMed1996

A doubly cross-linked human hemoglobin. Effects of cross-links between different subunits.

Human deoxyhemoglobin cross-linked with trimesyl tris(3,5-dibromosalicylate) produces the previously reported cross-linked hemoglobin in which the epsilon amino groups of the two beta chain 82 lysyl residues are joined by a trimesyl bridge. Further specific modification of this protein directed to the alpha subunits with bis(3,5-dibromosalicyl)fumarate gives a doubly cross-linked material in which the epsilon-amino groups of the two alpha chain 99 lysyl residues are now joined by a fumaryl bridge. The singly cross-linked beta chain species binds oxygen cooperatively with a high oxygen affinity (P50 = 4.8 torr at pH 7.4). The addition of the second cross-linking reduces the oxygen affinity to 15.9 torr, which compares with 13.0 torr for the singly cross-linked alpha chain species. The doubly cross-linked hemoglobin retains significant cooperativity with a Hill coefficient of 2.3 compared with 3.0 for unmodified hemoglobin. Because some of the groups responsible for the Bohr effect are acylated, this doubly cross-linked hemoglobin exhibits 25% of the normal Bohr effect and less than 20% of the normal chloride effect. The use of two distinct cross-links within the same tetramer provides a material for physical and structural analysis as well as for further modifications for specific applications. The results indicate that the cross-link introducing the lowest oxygen affinity in the two singly cross-linked species appears to control the overall affinity in this doubly cross-linked species.

Hemoglobins↗

PubMed1995

Monoclonal antibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium.

The monoclonal antibody 9EG7 has been previously found to recognize an epitope induced by manganese on the integrin beta 1 chain (Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9051-9055). Here we show that treatment of beta 1 integrins with manganese or soluble integrin ligands (e.g. fibronectin and RGD peptide) induced the 9EG7 epitope. This epitope was also induced upon EGTA treatment to remove calcium, and the addition of calcium inhibited 9EG7 epitope induction by manganese or by ligand. Further emphasizing the importance of the 9EG7 epitope, the 9EG7 antibody itself stimulated adhesion mediated by multiple beta 1 integrins, and conversely, ligands for alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, and alpha 5 beta 1 all stimulated 9EG7 expression. Together these results support a model whereby (i) calcium inhibits beta 1 integrin function because it prevents the appearance of a conformation favorable to ligand binding and (ii) manganese enhances beta 1 integrin function because it induces the same favorable conformation that is induced by adding ligand, or removing calcium. Notably, other beta 1-stimulating agents (magnesium and mAb TS2/16) did not induce 9EG7 expression unless ligand was also present. Thus, although 9EG7 may reliable detect the ligand-bound conformation of beta 1 integrins, its expression does not always correlate with integrin "activation". Finally, mouse/chicken beta 1 chimeric molecules were used to map the 9EG7 epitope to beta 1 residues 495-602 within the cysteine-rich region, and antibody cross-blocking studies showed that the 9EG7 epitope is distinct from all previously defined human beta 1 epitopes.

Amino Acid Sequence↗

PubMed1994

The chloride effect in human haemoglobin. A new kind of allosteric mechanism.

Chloride reduces the oxygen affinity of mammalian haemoglobin by acting as an allosteric effector that stabilizes the quaternary deoxy (T) structure. Perutz and others showed evidence that it does so by neutralizing electrostatic repulsion by an excess of positive charges in the cavity that runs through the centre of the molecule, but without binding to any specific site. On the basis of this proposal, any amino acid substitutions in the central cavity that halve the number of excess positive charges should halve the chloride effect, neutralization of the excess positive charges should inhibit it and introduction of additional positive charges should enhance it. Charge changes on the surface of the molecule should leave it unaltered. We have tested this proposal by measuring the chloride effects in several abnormal human haemoglobins with replacements of polar residues in the central cavity or on the surface that we happened to come across. They all proved consistent with the proposal. It appears that diffusible electrolytes can modify allosteric equilibria without necessarily binding to any specific site. Our proposal also implies that amino acid substitutions that make the central cavity more electropositive should destabilize the T-structure and therefore increase the oxygen affinity, while substitutions that make it more electronegative should do the reverse. A survey of all substitutions reported in the literature shows that this is true, with a few exceptions due to special stereochemical effects.

Allosteric Regulation↗

PubMed1994

Hemoglobin Denver [alpha 2 beta 2(41) (C7) Phe-->Ser]: a low-O2-affinity variant associated with chronic cyanosis and anemia.

OBJECTIVE: To report a previously undescribed low-O2-affinity hemoglobin variant that is associated with chronic cyanosis. DESIGN: Pertinent laboratory and historical data for the index case (from Denver, Colorado) and certain family members were recorded, and the hemoglobin variant was characterized. MATERIAL AND METHODS: Electrophoresis, high-performance liquid chromatography (HPLC), and isoelectric focusing were used to examine blood specimens for the presence of hemoglobin variants, and the O2 affinity of whole blood was determined. The abnormal peptide detected on reverse-phase HPLC of separated globin chains was analyzed for its amino acid composition and sequence. RESULTS: Although no abnormal hemoglobin band separated from hemoglobin A on electrophoresis, HPLC, and isoelectric focusing, a heat test showed hemoglobin instability, and O2 affinity studies disclosed an appreciably right-shifted dissociation curve. On chromatography, the new variant--hemoglobin Denver--was found to be due to a substitution of serine for phenylalanine at position 41 (C7) in the beta chain. In addition to substantial reduction in O2 affinity, hemoglobin Denver is accompanied by moderate reticulocytosis and mild anemia. CONCLUSION: Hemoglobin Denver causes no clinical symptoms other than cyanosis, which is attributable to the low O2 affinity.

Adolescent↗

PubMed1994

Hb Washtenaw [ beta 11(A8)Val-->Phe]: an electrophorectically silent, unstable, low oxygen affinity variant associated with anemia and chronic cyanosis.

Hb Washtenaw [beta 11(A8)Val-->Phe] is a new, low oxygen affinity variant with a previously undescribed substitution, identified in seven members over three generations of a Hungarian-American family. The hemoglobin is mildly unstable and the family members studied are clinically asymptomatic but mildly cyanotic, and some exhibit mild anemia. The index case had severe pulmonary hypertension and some of the family members had increased pulmonary vascular resistance on echocardiography. An association between the inheritance of this abnormal hemoglobin and the pathogenesis of primary pulmonary hypertension is suggested but the mechanism is unclear.

Adolescent↗

PubMed1994

Cross-linking hemoglobin by design: lessons from using molecular clamps.

The development of a red cell substitute by chemical modification of hemoglobin has been approached as a systematic, iterative process. Acyl phosphate methyl esters were designed as anionic electrophiles to permit selective acylation of amino groups in the cationic site of hemoglobin which binds polyanions. Kinetic studies with systematically substituted acyl phosphates and amines show that the reaction is controlled by a reversible addition step followed by an irreversible elimination step. Acyl phosphate methyl esters which are derivatives of rigid dicarboxylic acids introduce cross-links in human hemoglobin between amino groups in the beta subunits (epsilon-NH2-Lys-82, alpha-NH2-Val-1) and permit correlation of oxygen binding properties with cross-link structure. The data suggest that the cross-link maintains cooperativity while reducing overall oxygen affinity by lowering the affinity of the R form for oxygen rather than by perturbing the R,T equilibrium of native hemoglobin. Materials produced from deoxyhemoglobin with a cross-link between positions 1 and 82 of the two beta units have appropriate oxygen affinity for red cell substitutes. The use of a trifunctional cross-linker, trimesyl tris(methyl phosphate) selectively produces hemoglobin with the desired 1-82 connection in good yield. The reagent is readily prepared and the properties of this chemically modified hemoglobin are suitable for trial as a red cell substitute, closely resembling those of optimized materials produced by recombinant technology. Further work is producing new chemicals and providing structural information.

Binding Sites↗

PubMed1993

A mutagenic study of the allosteric linkage of His(HC3)146 beta in haemoglobin.

We have examined the contribution of His(HC3)146 beta to the alkaline Bohr effect of human haemoglobin (HbA) by replacing it with Gln, using site-directed mutagenesis, and studying the structural and functional consequences. Oxygen equilibrium curves of the mutant show that the effect of pH on the oxygen affinity, the alkaline Bohr effect, is half that of HbA in the presence of chloride ion and less than 10% in its absence. Crystallographic analysis shows that the mutation introduced only small structural changes localized to the site of substitution, proving that the replacement of the hydrogen bond between the ionizable side-chain of His146 beta and Asp94 beta by a hydrogen bond between the unionizable side-chain of Gln146 beta and the same aspartate is solely responsible for the reduction of the alkaline Bohr effect. Our data confirm that His(HC3)146 beta is predominantly responsible for the chloride-independent component of the alkaline Bohr effect which results from the breaking of the hydrogen bond between His(HC3)146 beta and Asp(FG1)94 beta accompanying the transition from the quaternary deoxy to oxy-structure.

Allosteric Regulation↗

PubMed1993

Modification of human hemoglobin with methyl acyl phosphates derived from dicarboxylic acids. Systematic relationships between cross-linked structure and oxygen-binding properties.

Human hemoglobin was reacted with five dicarboxylic acid bis(methyl phosphate) reagents under different ligand conditions. The bis(methyl phosphate) reagents tested were derived from fumaric, isophthalic, terephthalic, trans-stilbene-3,3'-dicarboxylic, and trans-stilbene-4,4'-dicarboxylic acids. These acyl phosphate mixed anhydrides are anionic electrophiles and will react with N-terminal amino and lysyl epsilon-amino groups to form amides. The major and many of the minor reaction products that result have been isolated and structurally characterized by globin chain and peptide analysis. Products which are not cross-linked, intrachain linked, and interchain singly and doubly cross-linked occur in proportions which depend upon the reaction conditions and reagent. Modifications of the beta chains were limited to the amino groups of beta 1Val, beta 82Lys, and, to a minor extent, beta 144Lys. In the case of the smaller reagents, the amino groups of alpha 1Val, alpha 99Lys, and, to a minor extent, alpha 139Lys were modified. The oxygen binding affinities of most of the major modified hemoglobins have been measured and are characterized by P50 values from about 1/2 to over 5 times that of unmodified human hemoglobin. Most show strong cooperativity with Hill coefficients (n) of 2.0 or greater. Several of the products that are cross-linked between the beta 1Val of one chain and the beta 82Lys of the other chain have oxygen affinities in a physiologically useful range for oxygen transport and delivery. An inverse linear correlation has been found between the log of P50 and bridging distances for the hemoglobins cross-linked between beta 1Val of one chain and the beta 82Lys of the other chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Structure/function analysis of the integrin beta 1 subunit by epitope mapping.

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti-chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.

3T3 Cells↗

PubMed1992

Three-point cross-linking: potential red cell substitutes from the reaction of trimesoyl tris(methyl phosphate) with hemoglobin.

The symmetrical trifunctional cross-linking reagent trimesoyl tris(methyl phosphate) (3), reacts selectively with amino groups (beta 1Val and beta 82Lys) in the diphosphoglycerate binding site of human hemoglobin A, producing cross-linked tetrameric species in good yield. A major species is triply linked, alpha alpha beta 1(82) greater than B beta 82, where B symbolizes benzene-1,3,5-tricarbonyl. Both this triply linked species and the doubly linked species, alpha alpha beta 1B beta 82, produced from deoxyhemoglobin have a considerably lower oxygen affinity than does native hemoglobin while maintaining a high degree of cooperativity (n50 = 2.4), making them potentially useful as red cell substitutes, in principle delivering twice as much oxygen as whole blood between pO2 = 100 and = 40 Torr. The yield of products indicates that triply and doubly linked species form in parallel so that there are independent routes to each. It is proposed that differences in routes are due to stereoisomerism about the amide bonds which form from reaction of the reagent with the protein.

Amino Acid Sequence↗

PubMed1992

Structure-function relationships in the low-affinity mutant haemoglobin Aalborg (Gly74 (E18)beta----Arg).

Haemoglobin Aalborg (Gly74 (E18)beta----Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates; it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)beta----Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2.8 A resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)beta protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)beta forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18 beta on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.

Amino Acid Sequence↗

PubMed1991

Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains.

Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.

Carbon Monoxide↗

PubMed1991

Was the loss of the D helix in alpha globin a functionally neutral mutation?

Proteins in the globin family are found in a variety of species from bacteria to man. From the many globin sequences known, evolutionary trees have been constructed showing that alpha and beta globins diverged from a common ancestor between 425 and 500 million years ago, after vertebrate species had appeared and roughly when sharks and bony vertebrates diverged. The alpha and beta globins assemble to form tetrameric haemoglobin, alpha 2 beta 2, which can switch between quaternary states having high and low oxygen affinity. This allows the protein to bind oxygen cooperatively and therefore efficiently transport oxygen from the lungs to respiring tissues. The alpha and beta globins have closely related tertiary structures, being alpha-helical proteins with similar haem-binding sites. Most globins consist of eight helices, designated A to H from the N terminus, connected by short nonhelical segments, but all known vertebrate alpha globins lack a D helix. Because the loss of this helix by alpha globin occurred shortly before tetrameric haemoglobin appeared, it might be a functionally important mutation required for a tetramer assembly or allostery. We have now tested this idea by engineering human haemoglobins containing beta subunits without a D helix and alpha subunits with a D helix. Both of these mutations have little effect on the oxygen-binding properties of the molecule. Thus it is possible that deletion of the D helix in the alpha subunit was caused by a neutral mutation.

Escherichia coli↗

PubMed1991

Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin.

We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.

Escherichia coli↗