Low oxygen affinity mutant hemoglobins Number:6,803,212 from the United States Patent and Trademark Office (PTO) owispatent Non-naturally occurring mutant hemoglobins rHb (.beta.N108Q) and rHb (.beta.L105W) are provided that have a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding. rHb (.beta.N108Q) also exhibits enhanced stability against autoxidation. The mutant hemoglobins are preferably produced by recombinant DNA techniques. Such mutant hemoglobins may be used as a component of a blood substitute and hemoglobin therapeutics.<H hemoglobins, affinity, Low, oxygen, affi, 6803212.html, Patent, pto, 6803212

Title: Low oxygen affinity mutant hemoglobins

Abstract: Non-naturally occurring mutant hemoglobins rHb (.beta.N108Q) and rHb (.beta.L105W) are provided that have a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding. rHb (.beta.N108Q) also exhibits enhanced stability against autoxidation. The mutant hemoglobins are preferably produced by recombinant DNA techniques. Such mutant hemoglobins may be used as a component of a blood substitute and hemoglobin therapeutics.

Patent Number: 6,803,212 Issued on 10/12/2004 to Ho, et al.

Inventors: Ho; Chien (Pittsburgh, PA); Tsai; Ching-Hsuan (Pittsburgh, PA); Fang; Tsuei-Yun (Yunlin, TW); Shen; Tong-Jian (Pittsburgh, PA)
Assignee: Carnegie Mellon University (Pittsburgh, PA)

Appl. No.: 09/986,633

Filed: November 9, 2001

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
598218	Jun., 2000	6486123

Current U.S. Class: 435/69.1 ; 435/252.3; 435/320.1; 435/325; 530/385; 536/23.1

Field of Search: 435/69.1,320.1,325,252.3 536/23.1 530/385

References Cited [Referenced By]

U.S. Patent Documents


5028588	July 1991	Hoffman et al.
5753465	May 1998	Ho et al.
5843888	December 1998	Ho et al.

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Primary Examiner: Carlson; Karen Cochrane
Attorney, Agent or Firm: Reed Smith LLP

Government Interests

The present invention was developed in part with government support under grant numbers HL-24525 and HL-58249. The government has certain rights in this invention.

Parent Case Text

This application is a divisional application of Ser. No. 09/598,218, filed Jun. 21, 2000, now U.S. Pat. No. 6,486,123.

Claims

What is claimed is:

1. Plasmid pHE7009.

2. A method of producing artificial hemoglobin, comprising: introducing an expression plasmid which contains a DNA coding sequence for human hemoglobin wherein the asparagine residue at position 108 of the .beta.-chains is replaced by a glutamine residue (SEQ ID NO: 8) into a suitable host other than an erythrocyte and growing the transformed cells; expressing said DNA to produce said artificial hemoglobin; and recovering and purifying said hemoglobin.

3. The method of claim 2, wherein said host cells are E. coli.

4. The method of claim 3, wherein said expression plasmid is pHE7009.

Description

FIELD OF THE INVENTION

This invention relates generally to novel mutant hemoglobins and more particularly relates to recombinant mutant hemoglobins "rHb (.beta.N108Q)" (alternative designation "rHb (.beta.108Asn.fwdarw.Gln)") and "rHb (.beta.L105W)" (alternative designation "rHb (.beta.105Leu.fwdarw.Trp") that possess low oxygen affinity, and high cooperativity in oxygen binding. In particular, rHb (.beta.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 .alpha. chains having 141 amino acid residues each and two identical .beta. 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.sub.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 .alpha..beta. 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 .alpha.- and .beta.-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.sub.max ") is a convenient measure of its oxygenation properties. See Dickerson, et al. (1983). Hb A has an n.sub.max value of approximately 3 in its binding with O.sub.2 under usual experimental conditions. Human abnormal Hbs with amino acid substitutions in the .alpha..sub.1.beta..sub.2 (or .alpha..sub.2.beta..sub.1) subunit interface generally result in high oxygen affinity and reduced cooperativity in O.sub.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 .alpha.94Asp and .beta.102Asn in the .alpha..sub.1.beta..sub.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 .alpha.94Asp position such as Hb Titusville (.alpha.94Asp.fwdarw.Asn) (Schneider, R. G., et al. Biochim. Biophys. Acta. 40:365 (1975), the disclosure of which is incorporated herein by reference) or the .beta.102Asn position such as Hb Kansas (.beta.102Asn.fwdarw.Thr) (Bonaventura, J., et al. J. Biol. Chem. 243:980 (1968), the disclosure of which is incorporated herein by reference), as well as others with mutations in the .alpha..sub.1.beta..sub.2 subunit interface, exhibit very low oxygen affinity. However, all these Hb mutants which directly disrupt the hydrogen bond between .alpha.94Asp and .beta.102Asn in the oxy form of Hb show greatly reduced cooperativity in the binding of oxygen and additionally dissociate easily into dimers when in the ligated state.

It has also been shown that during the transition from the deoxy-to the oxy-state, the .alpha..sub.1.beta..sub.2 subunit of Hb A undergoes a sliding movement, while the .alpha..sub.1.beta..sub.1 subunit interface remains nearly unchanged (See, Perutz, M. F. Nature 228: 726 (1970) ("Perutz (1970)"); Baldwin, J. M., et al. J. Mol. Biol. 129: 175 (1979); Baldwin, J. M., J. Mol. Biol. 136: 103 (1980); Shaanan, et al. (1983); and Fermi, G., et al. J. Mol. Biol. 175: 159 (1984), ("Fermi, et al., (1984)"), the disclosures of which are incorporated herein by reference. There are specific hydrogen bonds, salt bridges, and non-covalent interactions that characterize both subunit interfaces. The Hb molecule also has a lower oxygen affinity in the deoxy quaternary structure (T-structure) than in the oxy quaternary structure (R-structure) See, Dickerson, et al. (1983).

Low oxygen affinity human mutant Hbs which do not involve either .alpha.94Asp or .beta.102Asn also exist. For example, Hb Presbyterian (.beta.108Asn.fwdarw.Lys) (Moo-Penn, W. F., et al. FEBS Lett. 92:53 (1978) and O'Donnell, J. K., et al. J. Biol. Chem. 269:27692 (1994) (hereinafter "O'Donnell, et al. (1994)"); Hb Yoshizuka (.beta.108Asn.fwdarw.Asp), O'Donnell, et al. (1994) and recombinant Hb Mequon (.beta.41Phe.fwdarw.Tyr) (Baudin, V., et al. Biochim. Biophys. Acta. 1159:223 (1992), the disclosures of which are incorporated herein by reference, all exhibit low oxygen affinity compared to Hb A, but they all exhibit a variable amount of cooperativity as measured by the Hill coefficient, with n varying from 1.8 to 2.9. Tsai, C.-H., et al. Biochemistry 38:8751 (1999) (hereinafter, "Tsai, et al. (1999)") report Hb (.alpha.96Val.fwdarw.Trp, .beta.108Asn.fwdarw.Lys) which has low oxygen affinity and a greater tendency to switch to the T quaternary structure. Jeong, S. T., et al., Biochemistry 38:13433 (1999) (hereinafter, "Jeong, et al. (1999)") report that Hb (.alpha.29Leu.fwdarw.Phe, .alpha.96Val.fwdarw.Trp, .beta.108Asn.fwdarw.Lys) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Shen, et al. (1993) and U.S. Pat. No. 5,753,465, the disclosures of which are incorporated herein by reference, describe an E. coli expression plasmid (pHE2) in which synthetic human .alpha.- and .beta.-globin genes are coexpressed with the E. coli methionine aminopeptidase gene under the control of separate tac promotors. E. coli cells transformed with this plasmid express recombinant Hb A (hereinafter "rHb A") from which the N-terminal methionines have been effectively cleaved by the coexpressed methionine aminopeptidase. The resulting rHb A which lacks an N-terminal methionine is identical to the native Hb A in a number of structural and functional properties.

Kim, H.-W., et al. Proc. Natl. Acad. Sci. USA 91:11547 (1994) (hereinafter "Kim, et al. (1994)"), and U.S. Pat. No. 5,843,888, the disclosures of which are incorporated herein by reference, describe a non-naturally occurring mutant hemoglobin (rHb (.alpha.96Val.fwdarw.Trp) (alternative designation "rHb (.alpha.V96W)") that has a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding.

There remains a need, however, for additional mutant hemoglobin species that can be used as a component of a hemoglobin-based blood substitute or therapeutic agent. Of particular interest is a mutant hemoglobin that possesses low oxygen affinity, high cooperativity in oxygen binding, and increased stability against autoxidation. There is a further need for such a hemoglobin produced by recombinant methods and an efficient expression system for producing such a mutant hemoglobin in high yield, especially for use in a blood substitute product or hemoglobin therapeutics.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the present invention to provide mutant human hemoglobins with low oxygen affinity and high cooperativity in oxygen binding.

Another object of the present invention is to provide mutant hemoglobins with low oxygen affinity, high cooperativity in oxygen binding, and increased stability against autoxidation.

Another object of the present invention is to provide non-naturally occurring mutant human hemoglobins with low oxygen affinity and high cooperativity in oxygen binding.

Another object of the present invention is to provide non-naturally occurring mutant human hemoglobins with low oxygen affinity, high cooperativity in oxygen binding, and increased stability against autoxidation.

Another object of the present invention is to provide non-naturally occurring mutant human hemoglobins with low oxygen affinity, high cooperativity in oxygen binding, and preferably with stability against autoxidation that are produced artificially, preferably by recombinant means, and that have the correct heme conformation.

Another object of the present invention is to provide mutant hemoglobins that in a cell-free environment have similar oxygen binding properties as those of human normal adult hemoglobin in red blood cells.

Yet another object of the present invention is to provide mutant hemoglobins with low oxygen affinity and high cooperativity in oxygen binding in which the T-structure is stabilized while the R-structure is undisturbed.

Still another object of the present invention is to provide artificial hemoglobins for use as a hemoglobin-based oxygen carrier/red blood substitute or therapeutic agent.

These and other objects of the present invention are achieved by one or more of the following embodiments.

In one aspect, the invention features a non-naturally occurring mutant human hemoglobin wherein the asparagine residue at position 108 of the .beta.-chains is replaced by a glutamine residue.

In a preferred embodiment, the hemoglobin possesses low oxygen affinity as compared to human normal adult hemoglobin, high cooperativity in oxygen binding, increased stability against autoxidation, and is produced recombinantly.

In another aspect, the invention features an artificial mutant hemoglobin which in a cell-free environment has oxygen binding properties comparable to those of human normal adult hemoglobin in red blood cells wherein said hemoglobin contains a mutation such that the asparagine residue at position 108 of the .beta.-chains is glutamine.

A non-naturally occurring low oxygen affinity mutant hemoglobin with increased stability against autoxidation that has oxygen binding properties comparable to those of human normal adult hemoglobin in the presence of the allosteric effector 2,3-bisphosphoglycerate, wherein the asparagine residue at position 108 of each of the .beta.-chains is replaced by a glutamine residue.

In yet another aspect, the invention features a non-naturally occurring mutant human hemoglobin wherein the leucine residue at position 105 of the .beta.-chains is replaced by a tryptophan residue.

In a preferred embodiment, the hemoglobin possesses low oxygen affinity as compared to human normal adult hemoglobin, high cooperativity in oxygen binding, and is produced recombinantly.

In another aspect, the invention features an artificial mutant hemoglobin which in a cell-free environment has oxygen binding properties comparable to those of human normal adult hemoglobin in red blood cells wherein said hemoglobin contains a mutation such that the leucine residue at position 105 of the .beta.-chains is tryptophan.

A non-naturally occurring low oxygen affinity mutant hemoglobin that has oxygen binding properties comparable to those of human normal adult hemoglobin in the presence of the allosteric effector 2,3-bisphosphoglycerate, wherein the leucine residue at position 105 of each of the .beta.-chains is replaced by a tryptophan residue.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiment, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cDNA sequence (SEQ ID NO: 5) for the alpha- and beta-globin genes for rHb (.beta.N108Q) derived from plasmid pHE7009.

FIG. 1B is a cDNA sequence (SEQ ID NO: 7) for alpha- and beta-globin genes for rHb (.beta.L105W) derived from plasmid pHE7004.

FIGS. 2A and 2B show the FPLC profiles of rHb (.beta.N108Q) (peak b) (FIG. 2A) and rHb (.beta.L105W) (peak b) (FIG. 2B).

FIGS. 3A and 3B show the pH dependence of the oxygen affinity (P.sub.50) and the Hill coefficient (n.sub.max), respectively, of rHb (.alpha.L29F) (.quadrature.); rHb (.beta.N108Q) (.DELTA.); rHb (.alpha.L29F, .beta.N108Q) (X); rHb (.beta.L105W) (.tangle-solidup.); and Hb A (O) in 0.1 M sodium phosphate buffer at 29.degree. C. Oxygen dissociation data were obtained with 0.1 mM Hb.

FIG. 4 shows oxygen-binding curves of rHb (.beta.N08Q); rHb (.alpha.L29F, .beta.N108Q); rHb (.beta.L105W); and Hb A with and without the presence of allosteric effector, 5 mM 2,3-BPG, in 0.1 M phosphate buffer at pH 7.4 and 29.degree. C. Protein concentration was 0.1 mM heme.

FIG. 5 shows the autoxidation of Hb A (O); rHb (.beta.N108Q) (.tangle-solidup.); rHb (.beta.L105W) (.DELTA.); rHb (.alpha.V96W) (.gradient.); rHb (.alpha.V96W, .beta.N108K) (.tangle-soliddn.); rHb (.alpha.L29F, .beta.N108Q) (.diamond.); and rHb (.alpha.L29F, .alpha.V96W, .beta.N108K) (.quadrature.) in PlasmaLyte buffer in the presence of 5 mM EDTA and 5% D.sub.2 O at pH 7.4 and 37.degree. C., The autoxidation process was measured by monitoring the rate of disappearance of the oxy-marker at -2.34 ppm upfield from DSS by 300-MHz .sup.1 H-NMR.

FIGS. 6A and 6B are 500-MHz .sup.1 H-NMR spectra showing exchangeable proton resonances (FIG. 6A) and ring-current shifted proton resonances (FIG. 6B), respectively, of Hb A; rHb (.beta.N108Q), and rHb (.alpha.L29W, .beta.DL108Q), all in the CO form in 0.1 M sodium phosphate buffer at pH 7.0 and 29.degree. C.

FIGS. 7A and 7B are 300-MHz .sup.1 H-NMR spectra showing ferrous hyperfine-shifted N.sub..delta. H resonances of proximinal histidines and hyperfine-shifted and exchangeable proton resonances, respectively, of rHb A; rHb (.alpha.L29F); rHb (.beta.N108Q); and rHb (.alpha.L29W, .beta.N108Q), all in the CO form, in 0.1 M sodium phosphate buffer at pH 7.0 and 29.degree. C.

FIGS. 8A and 8B are 500-MHz spectra showing the exchangeable proton resonances of rHb (.beta.N108Q) in the CO form in 0.1 M sodium phosphate buffer at pH 7.0 at 500 MHz at various temperatures (7.degree. C., 11.degree. C., 17.degree. C., 23.degree. C., 29.degree. C.), without an allosteric effector (FIG. 8A) and with 4 mM inositol hexaphosphate ("IHP")(FIG. 8B).

FIGS. 9A and 9B are 500-MHz spectra showing the exchangeable proton resonances of rHb (.alpha.L29F, .beta.N108Q) in the CO form in 0.1 M sodium phosphate buffer at pH 7.0 at various temperatures (7.degree. C., 11.degree. C., 17.degree. C., 23.degree. C., 29.degree. C.), without (FIG 9A) and with (FIG. 9B) 4 mM IHP.

FIGS. 10A and 10B are 600-MHz .sup.1 H-NMR spectra showing exchangeable proton resonances (FIG. 10A) and ring-current shifted proton resonances (FIG. 10B) of 3-6% solutions of Hb A; rHb (.beta.L05W); rHb (.alpha.D94A, .beta.L105W); and rHb (.alpha.D94A) in the CO form in 0.1 M sodium phosphate at pH 7.0 and 29.degree. C.

FIGS. 11A and 11B are 600-MHz 2D heteronuclear multiple-quantum coherence ("HMQC") spectra of 5-8% solutions of .sup.15 N-labeled rHb (.beta.L105W) (FIG. 11A) and Hb A (FIG. 11B) in the CO form in 90% H.sub.2 O/10% D.sub.2 O in 0.1 M sodium phosphate at pH 7.0 and 29.degree. C.

FIGS. 12A-12D are 600-MHz 2D NOESY-HMQC ("NOESY"-nuclear Overhauser enhancement spectroscopy) spectra of a 5% solution of .sup.15 N-labeled rHb (.beta.L105W) in the CO form in 90% H.sub.2 O/10% D.sub.2 O in 0.1 M sodium phosphate at pH 7.0 and 29.degree. C. recorded at various mixing times: 15 ms (FIG. 12A); 30 ms (FIG. 12B); 60 ms (FIG. 12C); and 100 ms (FIG. 12D).

FIGS. 13A-13C are 300-MHz .sup.1 H-NMR spectra of 3-6% solutions of Hb A; rHb (.beta.L105W); rHb (.alpha.D94A, .beta.L105W); and rHb (rHb (.alpha.D94A) in the deoxy form in 0.1 M sodium phosphate at pH 7.0 and 29.degree. C. FIG. 13A shows hyperfine-shifted N.sub.67 H resonances of proximal histidines acquired at 300-MHz; FIG. 13B shows hyperfine shifted and exchangeable proton resonances acquired at 300-MHz; and FIG. 13C shows exchangeable proton resonances acquired at 300-MHz. Since rHb (.alpha.D94A, .beta.L105W) and rHb (.alpha.D94A) easily form met-Hb during the oxygenization process, a small amount of sodium dithionite was added to these NMR samples to diminish the formation of met-Hb.

FIG. 14 is a 600-MHz 2D HMQC spectrum of 5-8% solutions of .sup.15 N-labeled rHb (.beta.L105W) in the deoxy form in 90% H.sub.2 O/10% D.sub.2 O in 0.1 M sodium phosphate at pH 7.0 and 29.degree. C.

FIGS. 15A-15D are 600-MHz 2D NOESY-HMQC spectra of 5% solution of .sup.15 N-labeled rHb (.beta.L105W) in the deoxy form in 90% H.sub.2 O/10% D.sub.2 O in 0.1 M sodium phosphate at pH 7.0 and 29.degree. C. recorded at various mixing times: 15 ms (FIG. 15A); 30 ms (FIG. 15B); 60 ms (FIG. 15C); and 100 ms (FIG. 15D). The solid line between two cross peaks indicates the inter-residue NOE effect between the .sup.1 H.sub..epsilon.1 of one residue and the .sup.1 H.sub..delta.1 and .sup.1 H.sub..zeta.2 of the other residue for .beta.37Trp and .beta.105Trp.

FIGS. 16A-16B show exchangeable proton resonances in 600-MHz .sup.1 H-NMR spectra of 3-6% solutions of Hb A; rHb (.beta.L105W); rHb (.alpha.D94A, .beta.L105W); and rHb (.alpha.D94A) in the CO form in 0.1 M sodium phosphate at pH 7.0 and at 11.degree., 20.degree., and 29.degree. C. in the absence (FIG. 16A) and presence (FIG. 16B) of 2 mM IHP.

FIGS. 17A-17D are 600-MHz 2D heteronuclear single-quantum coherence ("HSQC") spectra of 5-8% solutions of .sup.15 N-labeled rHb (.beta.L105W) in the CO form in 90% H.sub.2 O/10% D.sub.2 O in 0.1 M sodium phosphate at pH 7.0 and various temperatures in the absence and presence of 2 mM IHP. FIG. 17A--29.degree. C. in the absence of IHP; FIG. 17B--at 29.degree. C. in the presence of 2 mM IHP; FIG. 17C--at 20.degree. C. in the presence of 2 mM IHP; and FIG. 19D--11.degree. C. in the presence of 2 mM IHP.

FIGS. 18A-18B show ring-current shifted proton resonances in 600-MHz .sup.1 H-NMR spectra of 3-6% solutions of Hb A; rHb (.beta.L105W); rHb (.alpha.D94A, .beta.L105W); and rHb (.alpha.D94A) in the CO form in 0.1 M sodium phosphate at pH 7.0 and various temperatures in the absence (FIG. 18A) and presence (FIG. 18B) of 2 mM IHP.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, "Hb A" or "native Hb A" means human normal adult hemoglobin as obtained from human subjects.

"Recombinant human normal adult hemoglobin," "rHb A," and "unmodified rHb A" mean human normal adult hemoglobin produced through recombinant DNA technology and having essentially the same structure and function as native Hb A as described by Shen, et al. (1993), and U.S. Pat. No. 5,753,465.

"rHb (.beta.L105W)" refers to a recombinant mutant human hemoglobin in which the leucine residue at position 105 of each of the .beta.-chains of the Hb molecule has been replaced by a tryptophan residue. This hemoglobin possesses low oxygen affinity and high cooperativity in oxygen binding compared to Hb A. rHb (.beta.L105W) is designed to form a hydrogen bond between .alpha.94Asp and .beta.105Trp in the .alpha..sub.1.beta..sub.2 subunit interface in order to lower the oxygen affinity by stabilizing its deoxy quaternary structure.

"rHb (.beta.N108Q)" refers to a mutant human hemoglobin produced through recombinant DNA technology in which the asparagine residue at position 108 of the .beta.-chains located in the .alpha..sub.1.beta..sub.2 interface and in the central cavity of the Hb molecule, have been replaced by glutamine residues. This hemoglobin possesses low oxygen affinity, and high cooperativity in oxygen binding, and also increased resistance to autoxidation as compared to other known recombinant low oxygen affinity mutant hemoglobins, such as, rHb (.alpha.V96W) and rHb (.alpha.V96W, .beta.N108K).

"Autoxidation" refers to the turning or conversion of oxyhemoglobin ("HbO.sub.2 " or "oxy-Hb") into methemoglobin ("met-Hb"). In HbO.sub.2 the heme-iron atoms are in the reduced ferrous (Fe.sup.2+) state, however, in met-Hb, the heme-iron atoms are in the oxidized ferric (Fe.sup.3+) state.

"Deoxy" and "oxy" refer to the oxygenation state of the heme-iron atom in Hb A and rHbs. Oxyhemoglobin ("oxy-Hb" or "HbO.sub.2 ") has four oxygen molecules bound to the heme groups; deoxyhemoglobin ("deoxy-Hb") contains no oxygen molecules. In normal arterial blood, normal adult hemoglobin A ("Hb A") is in the oxy form ("Hb O.sub.2 A" or "oxy-Hb A"). In venous blood, a portion of Hb A is in the deoxy form ("deoxy-Hb A").

"Carbonmonoxy-Hb," "HbCO A," "rHbCO," and "CO form" all refer to hemoglobin bound to carbon monoxide molecules rather than oxygen molecules.

"Ferri-hemoglobin," "ferri-Hb," "ferric form," "methemoglobin," "met-Hb", and "Fe.sup.+3 state" all refer to hemoglobin with their respective heme-iron atoms oxidized to the ferric (Fe.sup.3+) state. Ferri-Hb does not bind oxygen.

"Methionine aminopeptidase" refers to the enzyme methionine aminopeptidase which specifically cleaves the amino-(N) terminal methionine residue from a peptide sequence.

"Oxygen affinity" means the strength of binding of oxygen to a hemoglobin molecule. High oxygen affinity means hemoglobin does not readily release its bound oxygen molecules. The P.sub.50 is a measure of oxygen affinity.

"Cooperativity" refers to the binding of oxygen by the four subunits of the hemoglobin molecule and is measured by the Hill coefficient (n.sub.max). For Hb A in 0.1 M sodium phosphate at pH 7.4 and 29.degree. C., n.sub.max is about 3.2.

The two classical quaternary structures are the T (tense) quaternary D structure for the low affinity deoxy-Hb and the R (relax) quaternary structure for the high affinity oxy-Hb. "R-type" or "R-like," and similar terms refer to those hemoglobins which exhibit characteristic quaternary structural markers, such as the proton resonance at 10.7 ppm from DSS on .sup.1 H-NMR spectra. "T-type" or "T-like" and similar terms refer to those hemoglobins which exhibit characteristic T quaternary structures, such as the proton resonance at .about.14.0 ppm from DSS on .sup.1 H-NMR spectra.

II. Methods and Results

Using the Escherichia coli expression system described by Shen, et al. (1993); U.S. Pat. No. 5,753,465; and Kim, et al. (1995); U.S. Pat. No. 5,843,888, new non-naturally occurring artificial recombinant hemoglobins ("rHbs") have been constructed, having low oxygen affinity while maintaining high cooperativity in oxygen binding. One of the rHbs, rHb (.beta.N108Q) also exhibits increased resistance to autoxidation as compared to certain other known low oxygen affinity mutants. More particularly, the present invention is directed to: a recombinantly produced mutant of Hb A, denoted herein as rHb (.beta.N108Q), in which the asparagine residues at position 108 of each of the .beta.-chains (SEQ ID NO: 8), located in the .alpha..sub.1.beta..sub.1 subunit interface and in the central cavity of the Hb molecule, have been replaced by glutamine residue; and a recombinantly produced mutant of Hb A, denoted herein as rHb (.beta.L105W) in which the leucine residues at position 105 of each of the .beta. chains (SEQ ID NO: 8) have been replaced by tryptophan and in this molecule a new hydrogen bond is formed from .beta.105Trp to .beta.94Asp in the .alpha..sub.1.beta..sub.1 subunit interface in order to lower the oxygen binding affinity by stabilizing its deoxy quaternary structure.

These new artificial hemoglobins, i.e., derived entirely from sources other than blood, possess a low oxygen affinity and high cooperativity in oxygen binding. Additionally, rHb (.beta.N108Q) exhibits increased resistance to autoxidation as compared to other known low oxygen affinity mutants, such as rHb (.alpha.V96W) and rHb (.alpha.V96W, .beta.N108K). Further, these new artificial hemoglobins exhibit no unused subunit disassociation when ligated. In a cell-free environment the rHbs of the present invention have similar or lower oxygen binding properties to those of Hb A in red blood cells. Such rhbs therefore are of value as hemoglobin-based oxygen carriers, i.e., potential blood substitutes, or hemoglobin therapeutics.

It is also within the scope of the present invention to prepare and use other low oxygen affinity hemoglobins with other appropriate mutations. In particular, the methods of the present invention may be used to produce other mutant hemoglobins with additional advantageous properties. Methods for evaluating the suitability of other useful mutants possessing the properties of such low oxygen affinity, high cooperativity, and increased resistance to autoxidation for use in a blood substitute or therapy are described herein below. The preferred materials and methods for obtaining rHb (.beta.L105W) and rHb (.beta.N108Q) are given in the following reference examples. While the rHbs of the present invention are preferably produced recombinantly, it is understood that non-recombinant methods may also be used.

The preferred mutant rHbs of the present invention, rHb (.beta.L105W) and rHb (.beta.N108Q), can switch from the R quaternary structure to the T quaternary structure in their ligated state upon the addition of an allosteric effector, IHP, and/or by lowering the temperature. The recombinant hemoglobins of the present invention can therefore be used to gain new insight regarding the nature of subunit interactions in the .alpha..sub.1.beta..sub.2 and .alpha..sub.1.beta..sub.1 interfaces and the molecular basis for the allosteric mechanism of hemoglobin.

As will be shown below, rHb (.beta.N108Q) of the present invention shows a low oxygen affinity, an enhanced Bohr effect, but a similar cooperativity as that of Hb A, and also exhibits slower autoxidation to methemoglobin ("met-Hb") as compared to other known low oxygen affinity recombinant hemoglobins such as, for example, rHb (.alpha.96Val.fwdarw.Trp) and rHb (.alpha.96Val.fwdarw.Trp, .beta.108Asn.fwdarw.Lys) (Kim, H.-W., et al. Biochemistry 35:6620-6627 (1996) (hereinafter "Kim, et al. (1996)"); Ho, C., et al. Blood Substitutes: Present and Future Perspectives of Blood Substitutes (Tsuchida, E., Ed.), Elsevier Science SA, Lausanne, Switzerland, pp. 281-296 (1998) (hereinafter "Ho, et al. (1998)"); Jeong, et al. (1999); and Tsai, et al. (1999), the disclosures of which are incorporated herein by reference), oxidize much faster. Therefore, rHb (.beta.N108Q) can be useful for hemoglobin-based oxygen carriers and hemoglobin therapeutics.

Proton nuclear magnetic resonance (".sup.1 H-NMR") studies show that rHb (.beta.N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the .alpha..sub.1.beta..sub.1 and .alpha..sub.1.beta..sub.2 subunit interfaces as compared to those of Hb A. .sup.1 H-NMR studies also demonstrate that rHb (.beta.N108Q) can switch from the R quaternary structure to the T quaternary structure without changing its ligation state upon the addition of an allosteric effector, IHP, and/or by lowering the temperature. This suggests that the T quaternary structure of rHb (.beta.N108Q) is more stable than that of Hb A. This is consistent with the molecular mechanism of low-oxygen affinity found in rHb (.alpha.V96W) (Kim, H.-W., et al., J. Mol. Biol. 248:867 (1995) (hereinafter "Kin, et al. (1995)"); U.S. Pat. No. 5,843,888) and rHb (.alpha.V96W, .beta.N108Q) (Ho, et al. (1998); Tsai, et al. (1999)).

It has been reported by Carver, T. E., et al. J. Biol. Chem. 267: 14443 (1992); Brantley, R. E. Jr., et al. J. Biol. Chem. 268: 6995 (1993) (hereinafter "Brantley, et al. (1993)"; and Eich, R. F., et al. Biochemistry 35: 6976 (1996), the disclosures of which are incorporated herein by reference, that substitution of the Leu residue for phenylalanine at the B10 position can inhibit autoxidation in myoglobin and that at the B10 position of the .alpha.-chain can lower NO reaction with deoxy- and oxy-Hb A. Reduction of the NO reaction with oxy-Hb A by appropriate mutations, i.e., .alpha.L29F, in the distal heme pocket has been associated with reduction of the hypertensive effect recorded in vivo (Doherty, D. H., et al. Nature Biotech. 16: 672 (1998), the disclosure of which is incorporated herein by reference). Hence, as detailed below, such mutation was further introduced into .beta.N108Q to produce a double mutant, rHb (.alpha.L29F, .beta.N108Q). It was found that this double mutant is more stable against autoxidation as compared to rHb (.beta.N108Q), but exhibits comparable oxygen binding properties to those of Hb A in the presence of allosteric effector, 2 mM 2,3-BPG.

Mutant rHb (.beta.L105W) was designed to form a new hydrogen bond from .beta.105Trp to .alpha.94Asp in the .alpha..sub.1.beta..sub.2 subunit interface in order to lower the oxygen binding affinity by stabilizing its deoxy quaternary structure. It was found that rHb (.beta.L105W) possesses a very low oxygen affinity and maintains high cooperativity (P.sub.50 =28.2 mm Hg, n.sub.max =2.6 in 0.1 M sodium phosphate at pH 7.4 and 29.degree. C.) as compared to Hb A (P.sub.50 =9.9 mm Hg, n.sub.max =3.2 in 0.1 M sodium phosphate at pH 7.4 and 29.degree. C.). Mutant rHb (.alpha.D94A, .beta.L105W) and rHb (.alpha.D94A) were designed to provide evidence that rHb (.beta.L105W) forms a new hydrogen bond from .beta.105Trp to .alpha.94Asp in the .alpha..sub.1.beta..sub.2 subunit interface of the deoxy quaternary structure. The multinuclear, multidimensional nuclear magnetic resonance ("NMR") studies performed in accordance with the present invention in .sup.15 N-labeled rHb (.beta.L105W) have identified the resonance of the indole nitrogen-attached proton of .beta.105Trp for rHb (.beta.L105W). .sup.1 H-NMR studies on Hb A and mutant rHbs were used to investigate the structural basis for the low oxygen affinity of rHb (.beta.L105W). NMR results show that rHb (.beta.L105W) forms a new hydrogen bond from .beta.105Trp to .alpha.94Asp in the .alpha..sub.1.beta..sub.2 subunit of the deoxy quaternary structure. It is believed that the low oxygen affinity of rHb (.beta.L105W) is due to the formation of a new hydrogen bond between .beta.105Trp and .alpha.94Asp in the deoxy quaternary structure.

Proton nuclear magnetic resonance ("NMR") spectroscopy was used to study the tertiary and quaternary structures of Hbs in solution (Ho, et al. (1992)). Several exchangeable proton resonances at .about.15 to .about.9 ppm from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate ("DSS") have been characterized as intersubunit H-bonds in the .alpha..sub.1.beta..sub.1 and .alpha..sub.1.beta..sub.2 subunit interfaces in both oxy and deoxy states of Hb A. These H-bonded protons observed by NMR can be used as structural markers in functional studies. In particular, the resonance at .about.14 ppm from DSS has been identified as the inter-subunit H-bond between .alpha.42Tyr and .beta.99Asp in the .alpha..sub.1.beta..sub.2 interface of deoxy-Hb A, a characteristic feature of the T-structure of Hb A (Fung, L. W.-M., et al. Biochemistry 14:2526 (1975) (hereinafter "Fung, et al. (1975)"), 1975; Russu, I. M., et al. Biochem. Biophys. Acta 914:40 (1987) (hereinafter "Russu, et al. (1987)"). By observing this T-structure marker in both the deoxy and the CO forms of Hbs under various conditions, the stability of the T-conformation can be assessed and the transition from the T- to the R-structure can be monitored.

In the present invention, the strategy for designing rHbs with low oxygen affinity and high cooperativity was to stabilize the T-structure while not perturbing the R-structure. (See, Ho, et al. (1998); Tsai, et al. (1999)). This strategy has been demonstrated in the design of rHb (.alpha.V96W), which has low oxygen affinity and normal cooperativity (Kim et al. (1995); U.S. Pat. No. 5,843,888). This designed mutation is located at the .alpha..sub.1.beta..sub.2 subunit interface and in the central cavity of the Hb molecule. According to .sup.1 H-NMR studies, rHbCO (.alpha.V96W) can switch from the R-structure to the T-structure without changing its ligation state when the temperature is lowered and/or when IHP, an allosteric effector, is added. The crystal structure of rHb (.alpha.L96W) in its T-state has shown a novel water-mediated H-bond between .alpha.96Trp N.epsilon..sub.1 and .beta.101Glu O.epsilon..sub.2 in the .alpha..sub.1.beta..sub.2 subunit interface (Puius, T. A., et al. Biochemistry 37: 9258 (1998) (hereinafter "Puius", et al. (1998)"). Both .sup.1 H-NMR studies and the crystal structure indicate that the T-structure of this rHb is stabilized. In the present invention, the NMR studies have also shown that rHbCO (.beta.N108Q) and rHbCO (.beta.N105W) can switch to the T quaternary structure even when they are still ligated. These results suggest that the T structure of these two rHbs are more stable than that of Hb A.

As stated above, the methods of the present invention may also be used to produce other mutant artificial hemoglobins with different properties as well as hemoglobins with mutations that compensate for mutants that are naturally occurring. The preferred materials and methods for obtaining rHb (.beta.N108Q) and rHb (.beta.L105W) are given in the following reference example. Non-recombinant methods may also be used.

REFERENCE EXAMPLE

Construction of Expression Plasmids for rHb (.beta.N108Q) and rHb (.beta.L105W)

The E. coli Hb A expression plasmids pHE2 and pHE7, which respectively contain human .alpha.- and .beta.-globin genes and cDNAs, were used as the starting plasmids for expressing the mutant hemoglobins of the present invention. The construction of plasmids pHE2 and pHE7 and properties of the rHb A produced thereby are fully described in Shen, et al. (1993), U.S. Pat. No. 5,753,465, and Shen, T.-J., et al. Protein Eng. 10: 1085 (1997) (hereinafter "Shen, et al. (1997)"), Kim, et al. (1994), and U.S. Pat. No. 5,843,888), the disclosure of which is incorporated herein by reference.

The construction of plasmid pHE2009 for expression of mutant r Hb (.beta.N108Q) using synthetic globin genes was carried out as follows. The plasmid pHE2 was used as the starting plasmid and an oligonucleotide of sequence 5'-CGTCTGCTGGGTCAGGTACTAGTTTGCG-3'(SEQ ID NO: 1) (mutated codon is underlined) was purchased from DNA International, Inc. (Lake Oswego, Oreg.) and used as a primer to introduce the mutation .beta.N108Q into pHE2. Techniques for oligonucleotide synthesis are well known and this invention is not limited to any particular technique. The site-directed mutagenesis procedure followed the protocol of an "Altered Sites II In-Vitro Mutagenesis System" kit (Promega Corporation, Madison, Wis.) and the resultant plasmid pHE2009 contained the expected mutation .beta.N108Q.

The construction of plasmids pHE2020 (mutant rHb (.alpha.D94A) and pHE2004 (mutant rHb .beta.L105W)) using synthetic globin genes was similar to that of pHE2009, except the mutation oligonucleotide 5'-CTGCGTGTT GCTCCGGTCAACTTAAACTG-3' (SEQ ID NO: 2, mutated codon .alpha.D94A is underlined) and 5'-GGAAAACTTCCGATGGCTGGGTAACGTAC-3' (SEQ ID NO: 3, mutated codon .beta.L105W is underlined) were used. Both oligonucleotides were purchased from DNA International, Inc. (Lake Oswego, Oreg.).

The construction of plasmid pHE2017 (mutant rHb (.alpha.D94A, .beta.L105W)) was accomplished by ligating the 0.51-kb SmaI-PstI fragment of pHE2020 with the 6.34-kb kb PstI-SmaI fragment of pHE2004. The construction of plasmid pHE2018 for expression of mutant rHb (.alpha.L29F, .beta.N108Q) was accomplished by ligating the 6.06-kb PstI-BamHI fragment of pHE2009 with the 0.79-kb BamHI-PstI fragment of pHE284. The construction of plasmid pHE284 containing the mutation .alpha.L29F from plasmid pHE2 was reported previously by Jeong, et al. (1999).

The construction of plasmid pHE7009 for expression of mutant rHb (.beta.N108Q) using the human globin cDNAs was carried out as follows. The coding sequences of human .alpha.- and .beta.-globin cDNAs in plasmid pHE7 were inserted into pTZ18U (Bio-Rad Laboratories, Hercules, Calif.) by methods well known in the art. Site-directed mutagenesis was performed as described by Kunkel, T. M. et al., Proc. Natl. Acad. Sci. USA 82:488 (1985) the disclosures of which are incorporated herein by reference, and Shen, et al. (1993). An oligonucleotide of sequence 5'-ACAGACCAGTACTTGTCCCAGGAGCCT-3' (SEQ ID NO: 4) (mutated codon Asn.fwdarw.Gln is underlined) was purchased from DNA International, Inc. (Lake Oswego, Oreg.), and used as the mutation primer. The human normal .beta.-globin cDNA in plasmid pHE7 was then replaced with the mutated cDNA to produce plasmid pHE7009. The DNA sequences for the .alpha.- and .beta.-globin cDNAs in pHE7009 are shown in FIG. 1A (SEQ ID NO: 5). The amino acid sequence for the human beta chains of hemoglobin is shown in SEQ ID NO: 8. Plasmid pHE7009 in host cell E. coli JM109 and designated pHE7009/JM109 was deposited with the American Type Culture Collection of Manassas, Va. on Apr. 27, 2000 under number PTA-1768.

The construction of plasmid pHE7004 for expression of mutant rHb (.beta.N108Q) using the human globin cDNAs was carried out in the similar way as that of plasmid pHE7009, except an oligonucleotide of sequence 5'-CCTGAGAACTTCAGGTGGCTAGGCAACG TGCTGGTC-3' ((SEQ ID NO: 6), mutated codon Leu.fwdarw.Trp is underlined) was purchased from DNA International, Inc. (Lake Oswego, Oreg.) and used as the mutation primer. The DNA sequences of the .alpha.- and .beta.-globin cDNAs in pHE7004 are shown in FIG. 1B (SEQ ID NO: 7). The amino acid sequence for the human beta chains of hemoglobin is shown in SEQ ID NO: 8. Plasmid pHE7004 in host cell E. coli JM109 and designated pHE7004/JM109 was deposited with the American Type Culture Collection of Manassas, Va. on Apr. 27, 2000 under number PTA-1769.

Growth of Cells

Plasmids pHE7009, and pHE7004 were individually transformed in E. coli strain JM109 (Promega, Madison, Wis.) by methods well known in the art. E. coli cells were grown in Terrific Broth ("TB") medium plus 100 .mu.g/mL ampicillin in a 10-liter Microferm fermenter (New Brunswick Scientific, Model BioFlo 3000) at 32.degree. C. until the optical density at 600 nm reached 10. TB medium contains 1.2% bactotryptone, 2.4% bactoyeast extract, 0.17M KH.sub.2 PO.sub.4, 0.072M K.sub.2 HPO.sub.4, and 1% glucose solution. Expression of rHbs was induced by adding isopropyl .beta.-thiogalactopyranoside (Sigma, St. Louis, Mo.) to a concentration of 0.1-0.4 mM. The culture was then supplemented with hemin (20-50 mg/liter) (Sigma) and the growth was continued for at least another 4 hr. The cells were harvested by centrifugation and stored frozen at -80.degree. C. until needed for purification. For details, refer to Shen, et al. (1993), and Shen, et al. (1997).

Although E. coli cells are presently preferred for expressing and producing the recombinant mutant hemoglobin of the present invention, the invention is not limited to E. coli cells. Other appropriate expression systems such as yeast, insect cells and transgenic animals such as pigs, sheep, and cows may also advantageously be used to express mutant hemoglobins. Plasmids pHE7009 and pHE7004 have been optimized for E. coli cells, but other expression systems may be advantageously used. The plasmids can also be constructed with human genes.

Isolation and Purification of rHbs

The recombinant hemoglobins obtained from cells transformed with plasmids pHE7009 and pHE7004 were purified as essentially described by Shen, et al. (1993), and Shen, et al. (1997). The frozen stored cell paste was put into lysis buffer (40 mM trisbase/1 mM benzamidine) (Sigma) at 3 ml/gm of cell paste). The cell lysis procedure was used to pass the cell paste through a high-pressure homogenizer (Model EmulsiFlex-C5, Avestin) 3 times. The lysate was then centrifuged at 4.degree. C. for 2.5 hours at 13,000 rpm in a Beckman centrifuge (Beckman JA14 rotor). The supernatant from the lysate was saturated with CO gas and left at 30.degree. C. overnight as described by Tsai, et al. (1999). The supernatant was then put through a Millipore Minitan Acrylic Ultrafiltration system to concentrate the protein. Polyethyleneimine (Sigma) was added to a final concentration of 0.5% to precipitate nucleic acids. After centrifugation, the sample was dialyzed against 20 mM Tris-HCl/0.5 mM triethylenetetraamine ("TETA") (Sigma) at pH 8.3 overnight with one or two changes of buffer. Throughout the above procedures, the sample was kept at 4.degree. C. and maintained in a CO atmosphere. Following the procedures of Shen, et al. (1993) and Shen, et al. (1997), the rHb fraction collected after passage through a Q-Sepharose Fast-Flow column (Pharmacia Anion Exchanger) Pharmacia was oxidized and reduced, and converted to the CO form. This Hb solution was then purified by eluting through a fast protein liquid chromatography ("FPLC") Mono-S column (Pharmacia Cation Exchanger, HR 16/10) with a gradient of 10 mM sodium phosphate in 0.1-0.5 mM ethylenediaminetetraacetic acid ("EDTA") at pH 6.8 (eluent A) and 20 mM sodium phosphate in 0.1-0.5 mM EDTA at pH 8.3 (eluent B).

rHb (.beta.N108Q) was eluted individually in two major peaks. FIG. 2A shows peak a and peak b for rHb (.beta.N108Q). FIG. 2B shows rHb (.beta.L105W) was eluted individually in three major peaks, peaks a, b, and c. rHbs collected from peak b in both cases contained less than 2% methionine at the amino-termini and with the correct molecular weight.

Mass Spectrometry

Hb samples subjected to mass spectrometry were dialyzed extensively against distilled H.sub.2 O and then lyophilized. Immediately before analysis, the samples were dissolved in water to a concentration of 125 pmol of Hb per .mu.l of H.sub.2 O (7.8 mg/ml). Aliquots of these solutions were then diluted to give a final concentration of 10 pmol/.mu.l of 50:50 water/acetonitrile containing 0.2% formic acid. Aliquots (10 .mu.l) of these final solutions were introduced into the electrospray ion source at 5 .mu.l/minute.

The electrospray ionization analyses were performed on a VG Quattro-BQ (Fisons Instruments, VG Biotech, Altrincham, U.K.), as described by Shen, et al. (1993). Automated cycles of Edman degradation were performed on an Applied Biosystems gas/liquid-phase sequencer (Model 470/900A) equipped with an on-line phenylthiohydantoin amino acid analyzer (Model 120A). These two analytical procedures were used to assess the quality of the rHbs. All rHbs used in this study had the correct molecular weights and contained less than 2% of methionine at the amino termini.

Oxygen-Binding Properties of rHbs

Oxygen dissociation curves of rHbs were measured by a Hemox-Analyzer (TCS Medical Products, Huntington Valley, Pa.) at 29.degree. C. as a function of pH. The concentration of Hb used was approximately 0.1 mM per heme. The methemoglobin ("met-Hb") reductase system described by Hayashi, A., et al. Biochem. Biophys. Acta 310:309 (1973), the disclosure of which is incorporated herein by reference, was used if needed to reduce the amount of met-Hb in the sample. A visible absorption spectrum of each sample was recorded immediately after oxygen equilibrium measurement, and the met-Hb content was estimated by using the extinction coefficients of Hb reported by Antonini, E., Physiol. Rev. 45:123 (1965), the disclosure of which is incorporated herein by reference. Oxygen equilibrium parameters were derived by fitting Adair equations to each equilibrium oxygen-binding curve by a nonlinear least-squares procedures. P.sub.50, a measure of oxygen affinity, was obtained at 50% saturation. The Hill coefficient (n.sub.max), a measurement of cooperativity, was determined from the maximum slope of the Hill plot by linear regression. n.sub.max was derived between 60% and 65% oxygen saturation. The accuracy of P.sub.50 measurements in mm Hg is .+-.5% and that for n.sub.max is .+-.7%.

.sup.1 H-NMR Spectroscopy Measurements of rHbs

.sup.1 H-NMR spectra of rHbs were obtained on Bruker AVANCE DRX-300, AVANCE DRX-500, and AVANCE DRX-600 NMR spectrometers that were operated at 300, 500, and 600 MHz, respectively, and at temperatures ranging from 10.degree. C.-36.degree. C. All of the Hb samples were placed in 0.1 M sodium phosphate buffer (in 100% H.sub.2 O ) at pH 7.0. The Hb concentration range was approximately 5% (.about.3 mM in terms of heme). The water signal was suppressed by using the "jump-and-return" pulse sequence as reported by Plateau, P., et al. J. Am. Chem. Soc. 104:7310 (1982) (hereinafter "Plateau, et al. (1982)"), the disclosure of which is incorporated herein by reference. Proton chemical shifts are referenced to the methyl proton resonance of the sodium salt of 2,2-dimethyl-2-silapentane-5 sulfonate ("DSS") indirectly by using the water signal, which signal occurs at 4.76 ppm downfield from that of DSS at 29.degree. C., as the internal reference.

Autoxidation of rHbs

The autoxidation of rHbs was recorded by monitoring the disappearing rate of the oxy-marker (-2.34 ppm from DSS) from Bruker AVANCE DRX-300 .sup.1 H-NMR spectra. The autoxidation reaction was carried out in PlasmaLyte buffer (Baxter) (5% D.sub.2 O ) with 5 mM EDTA at pH 7.4 and at 37.degree. C. The HbO.sub.2 concentration was 5% (.about.3 mM in terms of heme).

Functional Studies

Oxygen-binding Properties of rHbs

FIGS. 3A and 3B show the oxygen-binding measurements of rHb (.alpha.L29F), rHb (.beta.N108Q), rHb (.alpha.29F, .beta.N108Q), rHb (.beta.L105W), and Hb A in 0.1 M sodium phosphate buffer as a function of pH at 29.degree. C. rHb (.beta.N108Q) exhibits a significantly lower oxygen affinity as compared to that of Hb A over the pH range from pH 6.79 to pH 8.09. The oxygenation process of rHb (.beta.N108Q) is very cooperative with an n.sub.max value of about 2.7 to 3.1 depending on the pH, compared to about 3.2 for Hb A (FIG. 3B). On the other hand, the mutation at the .alpha.-chain B10 position, i.e., .alpha.L29F, increases the oxygen affinity and decreases the cooperativity. rHb (.alpha.L29F, .beta.N108Q) shows slightly higher P.sub.50 values as compared to those of Hb A at pH<7.4, suggesting that the effect of the mutations on the oxygen affinity is additive. rHb (.alpha.L29F, .beta.N108Q) preserves cooperativity in binding of oxygen with an n.sub.max value of 2.4 to 2.8 (FIG. 3B). rHb (.beta.L105W) exhibits very low oxygen affinity (about 2-3 times lower) and maintains normal cooperativity from pH 7.0 to 8.0 as compared to Hb A.

FIG. 4 shows that the oxygen affinities of rHb (.beta.N108Q) and also rHb (.beta.L105W) measured in the absence of 2,3-BPG are lower than that of Hb A in the presence of 5 mM 2,3-BPG, making them potential candidates for an oxygen carrier in a blood substitute system. FIGS. 3A and 3B also show that the alkaline Bohr effect (which, in Hb A, results in a decrease in oxygen affinity with a lowering of the pH) is enhanced in rHb (.beta.N108Q) and rHb (.alpha.L29F, .beta.N108Q) compared to Hb A.

Table 1 below compares the number of Bohr protons released upon oxygenation per heme calculated from the linkage equation .DELTA.H.sup.+ =-.delta.log P.sub.50 /.delta.pH. (Wyman, J., Adv. Protein Chem. 4:407 (1948) and Adv. Protein Chem. 19:233 (1964), (hereinafter "Wyman, J. (1948) and (1964)") the disclosures of which are incorporated herein by reference. Both rHb (.beta.N108Q) and rHb (.alpha.L29F, .beta.NI08Q) release more Bohr protons than Hb A.

TABLE 1 Bohr effect of Hb A, rHb (.beta.N108Q), rHb (.alpha.L29F, .beta.N108Q), and rHb (.beta.L105W) in 0.1 M sodium phosphate buffer at 29.degree. C. Hemoglobin .differential.log P.sub.50 /.differential.pH in 0.1 phosphate Hb A 0.48 (pH 6.79-8.00) rHb (.beta.N108Q) 0.56 (pH 6.79-8.09) rHb (.alpha.L29F, .beta.N108Q) 0.67 (pH 6.79-7.97) rHb (.beta.L105W) 0.55 (pH 7.00-8.00)

Autoxidation

The autoxidation process was monitored for oxy-Hb A, oxy-rHb (.beta.N108Q), oxy-rHb (.alpha.L29F, .beta.N108Q), oxy-rHb (.beta.L105W) and three other known low-oxygen affinity mutants, oxy-rHb (.alpha.V96W), oxy-rHb (.alpha.L96W, .beta.N108K) and oxy-rHb (.alpha.L29F, .alpha.V96W, .beta.N108K), by a 300-MHz NMR spectrometer. The resonance at -2.34 ppm upfield from DSS has been assigned to the .gamma..sub.2 -CH.sub.3 of E11Val of Hb A in the oxy form of Hb A (Dalvit, C., et al., Biochemistry 24:3398 (1985), the disclosure of which is incorporated herein by reference). Monitoring the rate of disappearance of the oxy-marker (-2.34 ppm from DSS) as a function of time allows for the determination of the autoxidation rate of the Hb samples. The results are shown in FIG. 5. The percentage of ferrous-Hb varies with time (t) mono-exponentially and the autoxidation rate constant can be obtained from: [ferrous-Hb].sub.t =[ferrous-b].sub.t=0 x exp (-k.sub.auto x t), where k.sub.auto is the autoxidation rate constant. The autoxidation rate constants of Hb A and r Hbs are listed in Table 2 below. At pH 7.4 and 37.degree. C. in PlasmaLyte buffer, rHb (.beta.N108Q), rHb (.beta.L105W), rHb (.alpha.V96W), and rHb (.alpha.L96W, .beta.N108K) autoxidized 2.8-, 8-, 4.4-, and 8-times faster than Hb A. rHb (.beta.N108Q) is shown be to more stable against autoxidation than other known low-oxygen affinity mutants developed in the laboratory; i.e., rHb (.alpha.V96W), rHb (.beta.L105W), and rHb (.alpha.V96W, .beta.N108K). The autoxidation rate is slowed down by introducing the mutation .alpha.L29F into rHb (.beta.N108Q) and rHb (.alpha.V96W, .beta.N108K). rHb (.alpha.L29F, .beta.NI08Q) and rHb (.alpha.L29F, .alpha.V96W, .beta.N108K) autoxidized 2.5- and 2.8-times slower than rHb (.beta.N108Q) and rHb (.alpha.V96W, .beta.N108K), respectively. Thus, the mutation .alpha.L29F is very effective in slowing down the autoxidation process as suggested by the results on myoglobin (Brantley, et al., (199