Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1998-08-24
2000-11-21
Guzo, David
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530361, 435 691, C07K 14805, A61K 3514
Patent
active
061505064
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention is directed to modified hemoglobin-like compounds, and more particularly to modified hemoglobin-like polypeptides and proteins. The present invention is directed also to methods of purifying such modified hemoglobin-like compounds.
Hemoglobin (referred to herein as "Hb") is the oxygen-carrying component of blood. Hemoglobin circulates through the bloodstream inside small enucleate cells called erythrocytes (red blood cells). Hemoglobin is a protein constructed from four associated polypeptide chains, and bearing prosthetic groups known as hemes. The erythrocyte helps maintain hemoglobin in its reduced, functional form. The heme iron atom is susceptible to oxidation, but may be reduced again by one of two enzyme systems within the erythrocyte, the cytochrome b.sub.5 and glutathione reduction systems.
Hemoglobin binds oxygen at a respiratory surface (skin, gills, trachea, lung, etc.) and transports the oxygen to inner tissues, where it is released and used for metabolism. In nature, low molecular weight hemoglobins (16-120 kilodaltons) tend to be enclosed in circulating red blood cells, while the larger polymeric hemoglobins circulate freely in the blood or hemolymph.
The structure of hemoglobin is well known as described in Bunn & Forget, eds., Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders Co., Philadelphia, Pa.: 1986) and Fermi & Perutz "Hemoglobin and Myoglobin," in Phillips and Richards, Atlas of Molecular Structures in Biology (Clarendon Press: 1981).
About 92% of normal adult human hemolysate is Hb A.sub.o (designated alpha.sub.2 beta.sub.2 because it comprises two alpha and two beta chains). In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form a stable alpha/beta dimer, two of which in turn associate to form the tetramer. The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges. The amino add sequences of the alpha and beta globin polypeptide chains of Hb A.sub.o are given in Table 1 of PCT Publication No. WO 93/09143. The wild-type alpha chain consists of 141 amino acids. The iron atom of the heme (ferroprotoporphyrin IX) group is bound covalently to the imidazole of His 87 (the "proximal histidine"). The wild-type beta chain is 146 residues long and heme is bound to it at His 92.
The human alpha and beta globin genes reside on chromosomes 16 and 11, respectively. Bunn and Forget, infra at 172. Both genes have been cloned and sequenced, Liebhaber, et al., PNAS 77: 7054-58 (1980) (alpha-globin genomic DNA); Marotta, et al., J. Biol. Chem., 252:5040-53 (1977) (beta globin cDNA); Lawn, et al., Cell, 21:647 (1980) (beta globin genomic DNA).
Hemoglobin exhibits cooperative binding of oxygen by the four subunits of the hemoglobin molecule (the two alpha globins and two beta globins in the case of Hb A.sub.o), and this cooperativity greatly facilitates efficient oxygen transport. Cooperativity, achieved by the so-called heme-heme interaction, allows hemoglobin to vary its affinity for oxygen. Cooperativity can also be determined using the oxygen dissociation curve (described below) and is generally reported as the Hill coefficient, "n" or "n.sub.max." Hemoglobin reversibly binds up to four moles of oxygen per mole of hemoglobin.
Oxygen-carrying compounds are frequently compared by means of a device known as an oxygen dissociation curve. This curve is obtained when, for a given oxygen carrier, oxygen saturation or content is graphed against the partial pressure of oxygen. For Hb, the percentage of saturation increases with partial pressure according to a sigmoidal relationship. The P.sub.50 is the partial pressure at which the oxygen-carrying species is half saturated with oxygen. It is thus a measure of oxygen-binding affinity; the higher the P.sub.50, the more readily oxygen is released.
The ability of hemoglobin to alter its oxygen affinity under physiological conditions, increasing the efficiency of oxygen transport around the body, is largely dependent
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Anderson David C.
Anthony-Cahill Spencer
Epp Janet K.
Kerwin Bruce A.
Madril Dominic G.
Baxter Biotech Technology Sarl
Guzo David
Shuman Jon
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