Low oxygen affinity mutant hemoglobins

Details Low oxygen affinity mutant hemoglobins Low oxygen affinity mutant hemoglobins

2001-11-09

2004-10-12

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...

C435S320100, C435S325000, C435S252300, C536S023100, C530S385000

Reexamination Certificate

active

06803212

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to novel mutant hemoglobins and more particularly relates to recombinant mutant hemoglobins “rHb (&bgr;N108Q)” (alternative designation “rHb (&bgr;108Asn→Gln)”) and “rHb (&bgr;L105W)” (alternative designation “rHb (&bgr;105Leu→Trp”) that possess low oxygen affinity, and high cooperativity in oxygen binding. In particular, rHb (&bgr;N108Q) exhibits increased resistance to autoxidation as compared to other known low oxygen affinity mutants. This invention further relates to the preparation of mutant hemoglobins using recombinant DNA technology that are useful as substitutes for red blood cells and for hemoglobin-based therapeutics.
BACKGROUND OF THE INVENTION
The prevalence of infectious agents such as HIV and hepatitis in red blood cells of human blood products coupled with blood shortages from lack of suitable donors has led to great interest in the development of red blood cell substitutes, particularly human hemoglobin (“Hb”) and its derivatives. Hemoglobin-based oxygen carriers are potential sources of blood substitutes during emergency medical situations. See for example, Winslow, R. M., et al.
Hemoglobin
-
Based Red Cell Substitutes
, Johns Hopkins University Press, Baltimore (1992) (hereinafter “Winslow, et al. (1992)”), the disclosure of which is incorporated herein by reference.
Hemoglobin is the oxygen-carrying component of blood, circulated through the blood stream inside erythrocytes (red blood cells). Human normal adult hemoglobin (“Hb A”) is a tetrameric protein with a molecular weight of about 64,500 containing two identical &agr; chains having 141 amino acid residues each and two identical &bgr; chains having 146 amino acid residues each, with each also bearing prosthetic groups known as hemes. The erythrocytes help maintain hemoglobin in its reduced, functional form. The heme-iron atom is susceptible to oxidation, but may be reduced again by one of two systems within the erythrocyte, the cytochrome b
5
, and glutathione reduction systems. For a review on hemoglobin, see Dickerson, R. E., et al.
Hemoglobin: Structure, Function, Evolution, and Pathology
, Benjamin/Cummings, Menlo Park, Calif. (1983) (hereinafter “Dickerson, et al. (1983)”), the disclosure of which is incorporated herein by reference.
The oxygenation process of Hb A is cooperative, i.e., the binding of the first oxygen molecule enhances the binding of the second, third, and fourth oxygen molecules. The oxygenation process is also regulated by interactions between individual amino acid residues and various solutes, known as heterotropic allosteric effectors. These effectors include ions or molecules such as hydrogen ion, chloride, carbon dioxide, inorganic phosphate, and organic polyanions, such as 2,3-bisphosphoglycerate (“2,3-BPG”) and inositol hexaphosphate (“IHP”).
Hemoglobin is able to alter its oxygen affinity, thereby increasing the efficiency of oxygen transport in the body, due to its dependence on the allosteric effector 2,3-BPG. 2,3-BPG is present within erythrocytes at a concentration that allows hemoglobin to release bound oxygen to tissues. In the absence of 2,3-BPG, hemoglobin binds oxygen very tightly and does not readily release its bound oxygen. The hb A molecule alone, were it to be introduced into a subject, would not be able to properly allow oxygen to be delivered to tissues in the body due to a lack of 2,3-BPG, which lowers the oxygen affinity of Hb, in the blood plasma. See Winslow, et al. (1992). Any Hbs designed to be functional as Hb-based oxygen carriers or hemoglobin therapeutics should be able to deliver oxygen efficiently, i.e., they should load and unload cooperatively as Hb A does inside red blood cells.
The use of cell-free solutions of hemoglobin as a potential oxygen-carrying red cell substitute has been investigated for a long time. See, for example, Mulder, A. G., et al.,
J. Cell Comp. Physiol
. 5:383 (1934), the disclosure of which is incorporated herein by reference. However, the use of unmodified cell-free human hemoglobin purified from red blood cells suffers from several limitations in addition to contamination and supply limitations noted above, namely, an increase in oxygen affinity due to loss of allosteric effectors, such as 2,3-BPG, and dissociation of Hb tetramers into a &agr;&bgr; dimers which are cleared by renal filtration and which can cause long-term kidney damage. See, for example, Bunn, H. F., et al.
J. Exp. Med
. 129:909 (1969), the disclosure of which is incorporated herein by reference.
Human globins and hemoglobins have been expressed in the following: transgenic mice, see, for example, Chada, K., et al.,
Nature
(
London
) 314:377 (1985) and Townes, T. M., et al.
EMBO J
. 4:1715 (1985), transgenic swine as described by Swanson, M. E., et al.
Bio/Technology
10:557 (1992), insect cell cultures as reported by Groebe, D. R., et al.,
Protein Expression and Purification
3:134 (1992), yeast as described by Wagenbach, M., et al.
Bio/Technology
9:57 (1991) and DeLiano, J. J., et al.
Proc. Natl. Acad. Sci. USA
90:918 (1993), and
Escherichia coli
(“
E. coli
”) as described by Hoffman, S. J., et al.
Proc. Natl. Acad. Sci. USA
87:8521 (1990), Hernan, R. A., et al.
Biochemistry
31:8619 (1992), and Shen, T.-J., et al.
Proc. Natl. Acad. Sci. USA
90:8108 (1993) (hereinafter “Shen, et al. (1993)”), all the disclosures of which are incorporated herein by reference. In many respects, the
E. coli
system is the best choice for such purposes because of its high expression efficiency and the ease of performing site-directed mutagenesis.
The natural N-terminal valine residues of Hb A are known to play important roles in regulating oxygen affinity, the Bohr effect, and interactions with allosteric effectors and anions as reported by Bunn, H. F., et al. eds.
Hemoglobin: Molecular, Genetic and Clinical Aspects
(W. B. Saunders, Co., Philadelphia, Pa.) pp. 37-60 (1986) (hereinafter “Bunn, et al. (1986)”), the disclosure of which is incorporated herein by reference. The extra methionine can alter the N-terminal conformation of the Hb molecule as reported by Kavanaugh, J. S., et al.
Biochemistry
31:8640 (1992), the disclosure of which is incorporated herein by reference. Hence, the oxygenation properties of Hb depend on the integrity of the N-terminal residue thereby mandating the removal of the extra methionine residues from the N-termini of both the &agr;- and &bgr;-globins of the expressed Hb before the
E. coli
system can be used effectively for the production of desired unmodified and mutant Hbs.
The cooperative oxygenation of Hb, as measured by the Hill coefficient (“n
max
”) is a convenient measure of its oxygenation properties. See Dickerson, et al. (1983). Hb A has an n
max
value of approximately 3 in its binding with O
2
under usual experimental conditions. Human abnormal Hbs with amino acid substitutions in the &agr;
1
&bgr;
2
(or &agr;
2
&bgr;
1
) subunit interface generally result in high oxygen affinity and reduced cooperativity in O
2
binding compared to Hb A. See, for example, Dickerson, et al. (1983); Bunn, et al (1986) and Perutz, M. F., et al.
Mechanisms of Cooperativity and Allosteric Regulation in Proteins
Cambridge University Press (1990), the disclosure of which is incorporated herein by reference.
Hb A in its oxy form (Hb A with oxygen molecules) has a characteristic hydrogen bond between &agr;94Asp and &bgr;102Asn in the &agr;
1
&bgr;
2
subunit interface as reported by Shaanan, B., et al.
J. Mol. Biol
. 171:31 (1983), the disclosure of which is incorporated herein by reference (hereinafter “Shaanan, et al. (1983)”). Human Hbs with an amino acid substitution at either the &agr;94Asp position such as Hb Titusville (&agr;94Asp→Asn) (Schneider, R. G., et al.
Biochim. Biophys. Acta
. 40:365 (1975), the disclosure of which is incorporated herein by reference) or the &bgr;102Asn position such as Hb Kansas (&bgr;102Asn→Thr) (Bonaventura, J., et al.
J. Biol. Chem
. 243:980 (1968), the disclosure of which is incorporated herein by reference)

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