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...
Reexamination Certificate
1998-04-13
2001-02-06
Guzo, David (Department: 1636)
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...
C514S006900, C530S400000, C530S402000, C530S417000
Reexamination Certificate
active
06184356
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Hoffman and Nagai, U.S. Ser. No. 07/194,338, filed May 10, 1988, now U.S. Pat. No. 5,028,588, presently owned by Somatogen, Inc., relates to the use of low oxygen affinity and other mutant hemoglobins as blood substitutes, and to the expression of alpha and beta globin in nonerythroid cells. Hoffman and Nagai, U.S. Ser. No. 07/443,950, filed Dec. 1, 1989, abandoned, discloses certain additional dicysteine hemoglobin mutants; it is a continuation-in-part of Ser. No. 07/194,33 now U.S. Pat. No. 5,028,588. Anderson, et al., HEMOGLOBINS AS DRUG DELIVERY AGENTS, Ser. No. 07/789,177, filed Nov. 8, 1991, now abandoned discloses use of conjugation of hemoglobins with drugs as a means for delivery of the drug to a patient.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to multimeric hemoglobin-like proteins composed of two or more pseudotetramers linked together either by genetic fusion or by chemical crosslinking.
2. Description of the Background Art
A. Structure and Function of Hemoglobin
Hemoglobin (Hgb) or 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 here 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.
The structure of hemoglobin is well known. We herewith incorporate by reference the entire text of Bunn and Forget, eds.,
Hemoglobin: Molecular, Genetic and Clinical Aspects
(W. B. Saunders Co., Philadelphia, Pa.: 1986) and of Fermi and Perutz “Hemoglobin and Myoglobin,” in Phillips and Richards,
Atlas of Molecular Structures in Biology
(Clarendon Press: 1981).
About 92% of the normal adult human hemolysate is Hgb A (designated alpha2 beta2, because it comprises two alpha and two beta chains). Other recognized hemoglobin species are Hgb A
2
(&agr;
2
&dgr;
2
), Hgb A
1a
, Hgb A
1b
, and Hgb A
1c
, as well as the rare species Hgb F (&agr;
2
gamma), Hgb Gower-1 (Zeta
2
epsilon
2
), Hgb Gower-2 (alpha
2
epsilon
2
), Hgb Portland (Zeta
2
gamma
2
), and Hgb H (beta
4
) and Hgb Bart (gamma
4
). They are distinguished from Hgb A by a different selection of polypeptide chains.
The primary structure of a polypeptide is defined by its amino acid sequence and by identification of any modifications of the side chains of the individual amino acids. The amino acid sequences of both the alpha and beta globin polypeptide chains of “normal” human hemoglobin is given in Table 1. Many mutant forms are also known; several mutants are identified in Table 400. 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. Apohemoglobin is the heme-free analogue of hemoglobin; it exists predominantly as the &agr;&bgr;-globin dimer.
Segments of polypeptide chains may be stabilized by folding into one of two common conformations, the alpha helix and the beta pleated sheet. In its native state, about 75% of the hemoglobin molecule is alpha-helical. Alpha-helical segments are separated by segments wherein the chain is less constrained. It is conventional to identify the alpha-helical segments of each chain by letters, e.g., the proximal histidine of the alpha chain is F8 (residue 8 of helix F). The non-helical segments are identified by letter pairs, indicating which helical segments they connect. Thus, nonhelical segment BC lies between helix B and helix C. In comparing two variants of a particular hemoglobin chain, it may be enlightening to attempt to align the helical segments when seeking to find structural homologies. For the amino acid sequence and helical residue notation for normal human hemoglobin A
o
alpha and beta chains, see Bunn and Forget, supra, and Table 1 herein.
The tertiary structure of the hemoglobin molecule refers to the steric relationships of amino acid residues that are far apart in the linear sequence, while quaternary structure refers to the way in which the subunits (chains) are packed together. The tertiary and quaternary structure of the hemoglobin molecule have been discerned by X-ray diffraction analysis of hemoglobin crystals, which allows one to calculate the three-dimensional positions of the very atoms of the molecule.
In its unoxygenated (“deoxy”, or “T” for “tense”) form, the subunits of hemoglobin A (alpha1, alpha2, beta1, and beta2) form a tetrahedron having a twofold axis of symmetry. The axis runs down a water-filled “central cavity”. The subunits interact with one another by means of Van der Waals forces, hydrogen bonds and by ionic interactions (or “salt bridges”). The alpha1beta1 and alpha2beta2 interfaces remain relatively fixed during oxygenation. In contrast, there is considerable flux at the alpha1beta2 (and alpha2beta1) interface. In its oxygenated (“oxy”, or “R” for “relaxed” form), the intersubunit distances are increased.
The tertiary and quaternary structures of native oxyhemoglobin and deoxyhemoglobin are sufficiently well known that almost all of the nonhydrogen atoms can be positioned with an accuracy of 0.5 Å or better. For human deoxyhemoglobin, see Fermi, et al., J. Mol. Biol., 175: 159 (1984), and for human oxyhemoglobin, see Shaanan, J. Mol. Biol., 171: 31 (1983), both incorporated by reference.
Normal hemoglobin has cysteines at beta 93 (F9), beta 112 (G14), and alpha 104 (G11). The latter two positions are deeply buried in both the oxy and deoxy states; they lie near the &agr;
1
&bgr;
1
interface. Beta 93, however, in the oxy form is reactive with sulfhydryl reagents.
Native human hemoglobin has been fully reconstituted from separated heme-free alpha and beta globin and from hemin. Preferably, heme is first added to the alpha-globin subunit. The heme-bound alpha globin is then complexed to the heme-free beta subunit. Finally, heme is added to the half-filled globin dimer, and tetrameric hemoglobin is obtained. Yip, et al., PNAS (USA), 74: 64-68 (1977).
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 (two alpha-globins and two beta-globins in the case of Hgb A), 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. Hemoglobin reversibly binds up to four moles of oxygen per mole of Hgb.
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 Hgb, the percentage of saturation increases with partial pressure according to a sigmoid relationship. The P
50
is the partial pressure at which the oxygen-carrying solution is half saturated with oxygen. It is thus a measure of oxygen-binding affinity; the higher the P
50
, the more loosely the oxygen is held.
When the oxygen dissociation curve of an oxygen-carrying solution is such that the P
50
is less than that for whole blood, it is said to be “left-shifted.”
The oxygen affinity of hemoglobin is lowered by the presence of 2,3-diphosphoglycerate (2,3-DPG), chloride. ions and hydrogen ions. Respiring tissue releases carbon dioxide into the blood and
Anderson David C.
Mathews Antony J.
Stetler Gary L.
Baxter Biotech Technology Sarl
Guzo David
Senniger Powers Leavitt & Roedel
Shuman Jon
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