Haem disorder in reconstituted human haemoglobin

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

Section: Resultsmentioning

confidence: 98%

Haem disorder in two myoglobins: comparison of reorientation rate

Bellelli

Foon

Ascoli

et al. 1987

“…Haem orientational disorder also occurs in human haemoglobin. Docherty & Brown (1982) have measured haem disorder in reconstituted haemoglobin A by the 'coupled oxidation' approach in which the haem is degraded to various possible biliverdin isomers (a, fi, y and d). By analysis of the proportions of the isomers they concluded that reconstituted haemoglobin contained both orientational isomers but with only 20% of the disordered form.…”

Section: Resultsmentioning

confidence: 99%

“…The phenomenon of haem orientational disorder is well established in respiratory carriers and may also extend to electron-transfer proteins (La Mar et al, 1981;Docherty & Brown, 1982) as well as enzymes (La Mar et al, 1980a). On the basis of high-resolution proton n.m.r.…”

Section: Introductionmentioning

confidence: 99%

Characterization of haem disorder by circular dichroism

Aojula¹,

Wilson²,

Drake³

1986

“…5%), and this has been fully discussed elsewhere (Docherty & Brown, 1982). Because of this small yield, it is necessary to examine the possibility that the apparent coupled oxidation of cobalt(II) oxyhaemoproteins to biliverdin might, in fact, be due to contamination either by free haem or 'by the normal iron haemoproteins.…”

Section: Preparation Of Reconstituted Haemoproteinsmentioning

confidence: 99%

“…Degradation of protein-free haem (in pyridine) does indeed produce all four isomers (Bonnett & McDonagh, 1973;O'Carra, 1975) the conformation of the protein in the neighbourhood of the haem pocket (Brown, 1976;Brown & Docherty, 1978;Brown et al, 1981) or, alternatively, the orientation of the haem within the haem pocket (Docherty & Brown, 1982 Dickinson & Chien, 1973;Yonetani et al, 1974), and the complex may also exist in the cobalt(III) state. In principle, therefore, it would seem possible that cobalt protoporphyrin IX could replace haem as a substrate for microsomal haem oxygenase and in coupled oxidation reactions.…”

mentioning

confidence: 99%

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin

Vernon¹,

Brown²

1984

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.