Methods of controlling beta dimer formation in hemoglobin

Details Methods of controlling beta dimer formation in hemoglobin Methods of controlling beta dimer formation in hemoglobin

1999-05-14

2001-01-09

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C530S385000

Reexamination Certificate

active

06171826

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to expression of recombinant hemoglobin, and more particularly to methods of controlling beta dimer formation during the recombinant expression of the beta subunit.
BACKGROUND OF THE INVENTION
It is not always practical or safe to transfuse a patient with donated blood. In these situations, use of a red blood cell substitute is desirable. When human blood is not available or the risk of transfusion is too great, plasma expanders can be administered. However, plasma expanders, such as colloid and crystalloid solutions, replace only blood volume, and not oxygen carrying capacity. In situations where blood is not available for transfusion, a red blood cell substitute that can transport oxygen in addition to providing replacement is desirable.
Hemogloblin has been identified as a desirable red blood cell substitute. Hemoglobin (also 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
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
o
(designated alpha
2
beta
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 acid sequences of the alpha and beta globin polypeptide chains of Hb A
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
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
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
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
50
, the more readily oxygen is released.
The production of recombinant hemoglobin particularly in
E.coli
can lead to multiple species of recombinant hemoglobin. For example, in producing recombinant beta globins in bacterial systems, particularly
E.coli
, the beta globin can be expressed as a mixture of monomeric and dimeric beta globins. Thus, a need exists to control the formation of dimeric beta globins. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention relates to methods of controlling beta dimer formation in a hemoglobin solution. The methods are accomplished by altering a metal binding site, particularly a nickel binding site, on a beta globin or globin-like polypeptide to prevent or reduce said beta dimer formation. The methods are particularly suitable in the recombinant production of hemoglobin. Preferably, the metal binding site is the histidine adjacent to the N-terminal amino acid of the beta globin or globin-like polypeptide. For example, the histidine can be substituted with leucine or alanine in order to control the formation of beta dimers.
The present invention is further directed to methods of producing stable, intramolecularly crosslinked beta dimers by adding Ni(II) and oxone to a hemoglobin solution. Such methods will produce stable dimers within the hemoglobin contained in the solution between globins containing histidine adjacent to the N-terminal residue (hereinafter also referred to as “His2“). The hemoglobin can be derived from any source, for example, those sources described in WO 95/24213, published on Sep. 14, 1995, and incorporated herein by reference.


REFERENCES:
patent: 5545727 (1996-08-01), Hoffman et al.
patent: 5563254 (1996-10-01), Hoffman et al.
patent: 5578564 (1996-11-01), Chiver et al.
patent: 5840851 (1998-11-01), Plomer et al.
patent: 93/09143 (1993-05-01), None
patent: 95/14038 (1995-05-01), None
patent: 95/24213 (1995-09-01), None
patent: 97/04110 (1997-02-01), None
patent: 98/05773 (1998-02-01), None
Russu, et al., A Proton Nuclear Magnetic Resonance Investigation of Histidyl Residues in Human Normal Adult Hemoglobin, Biochemistry, 1982, vol. 21, pp. 5031-5043.
Hirel, et al., Extent of N-terminal Methionine Excision FromEscherichia coliProteins is Governed by the Side-chain Length of the Penultimate Amino Acid, Proc. Natl, Acad. Sci., vol. 86, pp. 8247-8251, Nov. 1989.
Climent, et al., Derivatization of &ggr;-Glutamyl Semialdehyde Residues in Oxidized Proteins by Fluoresceinamine, Analytical Biochemistry, vol. 182, pp. 226-232, 1989.
Stadtman, et al., Metal-Catalyzed Oxidation of Proteins, The Journal of Biological Chemistry, vol. 266, No. 4, pp. 2005-2008, 1991.
Schöneich, et al., Iron-thiolate Induced Oxidation of Methionine to Methionine Sulfoxide in Small Model Peptides. Intramolecular Catalysis By Histidine, Biochimica et Biophysica Acta, vol. 1158, ppg. 307, 1993.
Shibayama, et al., Oxygen Equilibrium Properties of Nickel (II)—Iron (II Hybrid Hemoglobins Cross-Linked between 82&bgr;1 and 82&bgr;2 Lysyl Residues by Bis (3,5-dibromosalicyl) fumarate: Determination of the First Two-Step Microscopic Adair Constants for Human Hemoglobin, Biochemistry, vol. 34, pp. 4773-4780, 1995.
Brown, et al., Highly Specific Oxidative Cross-Linking of Proteins Mediated by a Nickel-Peptide Complex, Biochemistry, vol. 34, pp. 4733-4739, 1995.
Dumoulin, et al., Loss of Allosteric Behaviour in Recombinant Hemoglobin &agr;2&bgr;292 (F8) His→Ala: Restoration Upon Addition of Strong Effectors, FEBS Letters, vol. 374, pp. 39-42, 1995.
Levin, et al., Methionine Residues as Endogenous A

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