Characterization of haem disorder by circular dichroism

Aojula, Harmesh; Wilson, Michael T.; Drake, Alex F.

doi:10.1042/bj2370613

Cited by 60 publications

(57 citation statements)

References 16 publications

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“…Thus, it serves as a sensitive probe of correct heme pocket refolding. In the heme pocket, in the absence of amino acid substitutions a decrease in ellipticity has been attributed to the presence of inverted heme (33). The CD spectra of HbA and ␣rHbA have a similar ellipticity (Fig.…”

Section: Discussionmentioning

confidence: 67%

Assembly of Human Hemoglobin

Sanna

Razynska

Karavitis

et al. 1997

Journal of Biological Chemistry

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The ␣-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native ␤-chains. The functional and structural properties of the expressed ␣-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl ؊ and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the ␣ 1 ␤ 1 and ␣ 1 ␤ 2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed ␣-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the ␣-globin refolds and incorporates heme in the absence of the partner ␤-chain. Although the CO binding kinetics of recombinant ␣-chains were the same as that of native ␣-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant ␣-chain assumes alternating conformations in the absence of ␤-chain and indicate that the isolated ␣-chain exhibits a higher degree of conformational flexibility than the ␣-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed ␣-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.

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Section: Discussionmentioning

confidence: 67%

Assembly of Human Hemoglobin

Sanna

Razynska

Karavitis

et al. 1997

Journal of Biological Chemistry

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“…For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J.…”

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confidence: 99%

“…(La Mar et al, 1984) or c.d. spectroscopy (Aojula et al, 1986) and the measured half-time ranges from hours to days depending on the experimental conditions (for example, pH and temperature).To a first approximation it may be assumed that the prevalent pathway to achieve haem reorientation requires dissociation of the porphyrin from the protein, since the haem pocket appears to be too small to allow the haem to rotate freely; for this reason we compared the rate of haem reorientation in two myoglobins whose physicochemical properties, particularly with respect to haem affinity, differ widely: those from sperm whale and from Aplysia limacina; in fact myoglobin from Aplysia limacina …”

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confidence: 99%

Haem disorder in two myoglobins: comparison of reorientation rate

Bellelli

Foon

Ascoli

et al. 1987

Biochemical Journal

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The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237,[613][614][615][616] for sperm whale myoglobin. The time course of the c.d. transition is found to be approx. 10 times faster in Aplysia than in sperm whale myoglobin, a result which is in agreement with the known structural and physicochemical properties of the two myoglobins; furthermore, these results confirm that c.d. and n.m.r. data on haem orientation in haemoproteins reflect the same molecular phenomenon. INTRODUCTIONSeveral haemoproteins have been described to exist in two interconvertible conformations, differing in their n.m.r. properties. La Mar and collaborators (La Mar et al., 1978) have shown that the coexistence of conformers is related to the fact that the intrinsically asymmetric haem molecule can assume two orientations differing by 180°rotation around its a-y axis. Often one of the conformers is largely prevalent at equilibrium, and constitutes the 'native' (or ordered) conformation as detected by X-ray crystallography. In the case of sperm whale myoglobin the disordered form accounts for only 10% of the total haemoprotein in solution (La Mar et al., 1983), while in other cases the disordered form can be as populated as the ordered one, as in tuna fish myoglobin (Levy et al., 1985).Immediately after reconstitution obtained by mixing sperm whale apomyoglobin with either ferrous or ferric haem, the disordered form accounts for approx. 50 % of the total pigment; this population ratio changes with time and decays slowly towards its equilibrium value. The re-equilibration has been followed by means of n.m.r. (La Mar et al., 1984) or c.d. spectroscopy (Aojula et al., 1986) and the measured half-time ranges from hours to days depending on the experimental conditions (for example, pH and temperature).To a first approximation it may be assumed that the prevalent pathway to achieve haem reorientation requires dissociation of the porphyrin from the protein, since the haem pocket appears to be too small to allow the haem to rotate freely; for this reason we compared the rate of haem reorientation in two myoglobins whose physicochemical properties, particularly with respect to haem affinity, differ widely: those from sperm whale and from Aplysia limacina; in fact myoglobin from Aplysia limacina

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“…For the adult and fetal Hbs these levels of haem disorder are thought to represent the equilibrium position in i o. The extent and dynamics of haem disorder can be measured by NMR spectroscopy of the paramagnetically shifted haem peripheral methyl protons [2,3] and by CD spectroscopy of the haem Soret band [5,6,9]. Nonequilibrium levels of haem disorder in myoglobin and Hb relax to equilibrium levels on incubating the ferric Hbs at room temperature for 24 h [2,3].…”

Section: Introductionmentioning

confidence: 99%

“…However, haem disorder also occurs naturally at low levels in sperm-whale (Physeter macrocephalus) myoglobin [1], and at high levels in myoglobin from yellowfin-tuna (Thunnus albacarus) muscle [4]. Haem disorder appears to have negligible functional consequences in sperm-whale myoglobin [5,6] -a finding consistent with the minor effects of reconstituting myoglobins with haems containing altered porphyrin substituents [7]. Haem disorder in Hb has been reported to somewhat increase autoxidation rates, and to alter subunit-ligand affinities [3].…”

Section: Introductionmentioning

confidence: 99%

Haem disorder in recombinant- and reticulocyte-derived haemoglobins: evidence for stereoselective haem insertion in eukaryotes

Mathews

Brittain

2001

Biochemical Journal

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We have used NMR spectroscopy to measure haem disorder in adult human haemoglobin (HbA) obtained from mature erythrocyte cells and from yeast expressing recombinant HbA. Reticulocyte-derived HbA contained much higher levels of haem disorder (11 % α-and 28 % β-subunit disorder) than observed for HbA from mature erythrocytes (1.5 % α-and 8 % β-subunit disorder). Thus, unlike in itro combination of haem and apoHb, biosynthetic haem insertion is not random with respect to orientation, but appears to show stereoselectivity. Recombinant HbA isolated from yeast showed 32 % α-and 45 % β-subunit

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Characterization of haem disorder by circular dichroism

Cited by 60 publications

References 16 publications

Assembly of Human Hemoglobin

Assembly of Human Hemoglobin

Haem disorder in two myoglobins: comparison of reorientation rate

Haem disorder in recombinant- and reticulocyte-derived haemoglobins: evidence for stereoselective haem insertion in eukaryotes

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