Structure of extracellular hemoglobin from the brine shrimp Artemia salina.

1. Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and preparative gel electrophoresis in non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with Mr 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.     

Quaternary structure of erythrocruorin from the nematode Ascaris suum. Evidence for unsaturated haem-binding sites.

The quaternary structure of erythrocruorin from the nematode Ascaris suum was studied. The native protein had a sedimentation coefficient, at a protein concentration of 1 mg/ml, of 11.6 +/- 0.3 S and an Mr, as determined by sedimentation equilibrium, of 332,000 +/- 17,000. SDS/polyacrylamide-gel electrophoresis gave one band with a mobility corresponding to an Mr of 43,000 +/- 2000. The Mr of the polypeptide chain was determined to be 41,600 +/- 1,500 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. Cross-linking with glutaraldehyde followed by SDS/polyacrylamide-gel electrophoresis yielded a maximal number of eight bands. The haem content of Ascaris erythrocruorin was observed to vary from one preparation to another. This finding was shown to be due to non-realization of the full binding capacity for haem. By titration with haemin, the haem content was found to attain a maximal value of 2.86 +/- 0.14%, corresponding to a minimal Mr per haem group of 21,000 +/- 1,000. Our findings indicate that Ascaris suum erythrocruorin is composed of eight identical polypeptide chains, carrying two haem sites each.     

A disulfide-bonded trimer of myoglobin-like chains is the principal subunit of the extracellular hemoglobin of Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris (3900 kDa) consists of at least six different polypeptide chains: I through IV (16-19 kDa), V (31 kDa) and IV (37 kDa) (Vinogradov, S.N., Shlom, J.M., Hall, B.C., Kapp, O.H. and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155). SDS-polyacrylamide gel electrophoresis of the unreduced hemoglobin shows that chains II, III and IV form a disulfide-bonded 50 kDa subunit. This subunit was isolated by gel filtration of the hemoglobin on Sephacryl S-200 (a) at neutral pH in 0.1% SDS and (b) in 0.1 M sodium acetate buffer (pH 4.0); in the latter case it retains heme. The 50 kDa subunit obtained by method (b) was reduced and subjected to chromatofocusing on PBE 94 column: the elution pattern obtained with Polybuffer 74 (pH 4.5) and monitored at 280 nm, consisted of three peaks A, B and C; peaks A and B but not C, had absorbance at 410 nm. SDS-polyacrylamide gel electrophoresis showed that peaks A, B and C corresponded to chains II, IV and III, respectively. Amino acid analyses and N-terminal sequence determinations identified chain II as the whose primary structure had been determined (Garlick, R. and Riggs, A. (1982) J. Biol. Chem. 257, 9005-9015). Carbohydrate analysis of the native hemoglobin shows it to contain 2.0 +/- 0.5% carbohydrate consisting of mannose and N-acetylglucosamine in a mole ratio of about 9:1. The carbohydrate content of the 50 kDa subunit is 1.8 +/- 0.5%; it consists of mannose and N-acetylglucosamine in the same ratio and it appears to be associated with chain IV. Rabbit polyclonal antisera to 50 kDa subunit, prepared by method (a), and to the native hemoglobin were shown to cross-react with the hemoglobin and the 50 kDa subunit, respectively, by immunodiffusion. One of eight mouse monoclonal antibodies directed against the native hemoglobin reacted strongly with the 50 kDa subunit prepared by methods (a) and (b) in an enzyme-linked immunosorbent assay (ELISA). Another monoclonal antibody reacted strongly with the 50 kDa subunit obtained by method (b). Neither of the two hybridomas exhibited a strong reaction with any of the three constituent chains of the 50 kDa subunit. These results suggest that the unusual disulfide-bonded 50 kDa subunit, consisting of three myoglobin-like polypeptide chains of which only two have heme, is an integral part of the native Lumbricus hemoglobin molecule.     

Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme

Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.     

Origin of the anomalous Fe-CO stretching mode in the CO complex of Ascaris hemoglobin

We report an unusually high frequency (543 cm(-)(1)) for an Fe-CO stretching mode in the CO complex of Ascaris suum hemoglobin as compared to vertebrate hemoglobins in which the frequency of the Fe-CO mode is much lower. A second Fe-CO stretching mode in Ascaris hemoglobin is observed at 515 cm(-1). We propose that these two Fe-CO stretching modes arise from two protein conformers corresponding to interactions of the heme-bound CO with the B10-tyrosine or the E7-glutamine residues. This postulate is supported by spectra from the B10-Tyr --> Phe mutant in which the 543 cm(-1) line is absent. Thus, a strong polar interaction, such as hydrogen bonding, of the CO with the distal B10 tyrosine residue is the dominant factor that causes this anomalously high frequency. Strong hydrogen bonding between O(2) and distal residues in the oxy complex of Ascaris hemoglobin has been shown to result in a rigid structure, rendering an extremely low oxygen off rate [Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. London B 163, 206-214]. In contrast, the CO off rate in Ascaris hemoglobin is very similar to that in sperm whale myoglobin. The high CO off rate relative to that of O(2) in Ascaris hemoglobin is attributed to a rapid equilibrium between the two conformations of the protein in the CO adduct, with the off rate being determined by the conformer with the higher rate.     

Evidence for an active-inactive subunit complex in jack bean urease.

The structural subunit molecular weight of jack bean urease has been determined to be 30,400 on the basis of the chemical evidence presented. Each polypeptide chain has been shown to possess four types of sulfhydryl groups. In half of the subunits the sulfhydryl group required for catalytic activity is involved in a disulfide bond. This results in a catalytic subunit of 60,800 molecular weight composed of two polypeptide chains but having only one active site.     

Proteolytic studies on the structure of bovine von Willebrand factor

Bovine von Willebrand factor (vWF) was digested with protease I (P-I), a metalloprotease isolated from rattlesnake venom. Digestion of vWF for 24 h with P-I yielded a terminal digest consisting of an equimolar mixture of two major fragments (apparent Mr 250K and 200K). The 250-kilodalton (kDa) fragment consists of a 125-kDa chain from one subunit and a 45- and 78-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment consists of a 97-kDa chain from one subunit and a 35- and 61-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment binds to heparin, and the heparin binding domain is located on the 97-kDa polypeptide chain. This fragment also competes with labeled, native vWF for binding to formalin-fixed human platelets, with an IC50 of 12.5 micrograms/mL (65 nM). However, native vWF has an IC50 of 2.5 micrograms/mL, indicating that the affinity of the 200-kDa fragment for platelets is approximately one-fifth that of vWF. The 200-kDa fragment agglutinates platelets, but its agglutinating ability is only 5% that of the native molecule. Only the 200-kDa fragment is recognized by monoclonal antibodies 2 and H-9, which are directed against vWF and inhibit vWF binding to platelet glycoprotein Ib (GPIb). Immunological studies, using nine monoclonal antibodies directed against vWF, and the demonstration that the heparin and GPIb binding domains are located on only one fragment suggest that the two fragments are composed of different regions of the vWF subunit. Analysis of the P-I cleavage pattern suggests that all vWF subunits are not cleaved in the same fashion. The first cleavage on half of the subunits generates the 45-kDa terminal and 175-kDa intermediate digest products. The 175-kDa chain is again cleaved, producing the 97- and 78-kDa terminal polypeptide chains. However, the first cleavage of the other subunits generates the 35-kDa terminal and the 186-kDa intermediate digest product, which upon cleavage produces the 125- and 61-kDa terminal polypeptide chains. Immunological data support the asymmetric cleavage pattern. An epitope for a monoclonal antibody is present on both the 186- and 175-kDa intermediate digest products but is only found on one terminal digest fragment, the 78-kDa polypeptide chain, suggesting that the 186- and 175-kDa polypeptides are cleaved at different sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump.

A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.     

Inhibition of S-adenosylmethionine-linked methylation can lead to neurite extension in neuroblastoma cells

The hog sucrase—isomaltase complex is anchored to the small-intestinal brush border membrane, as in the rabbit, via a hydrophobic segment located in the N-terminal region of the isomaltase subunit. The immediate precursor of the ‘final’ sucrase—isomaltase (i.e., pro-sucrase—isomaltase as prepared from adult hogs whose pancreas had been disconnected from the duodenum) is an amphiphilic single polypeptide chain of M_r 260 000–265 000. Its N-terminal sequence is virtually identical with (not merely homologous to) the corresponding region of the isomaltase subunit of ‘final’ sucrase-isomaltase. This shows that the isomaltase portion of pro-sucrase—isomaltase in the N-terminal ‘half’ of the precursor polypeptide chain. Thus the succession of domains in pro-sucrase—isomaltase and its mode of anchoring in the membrane could be deduced. On this basis a likely mechanism of biosynthesis and insertion is proposed.     

Structural studies of a branchiopod crustacean (Lepidurus bilobatus) extracellular hemoglobin. Evidence for oxygen-binding domains.

The extracellular hemoglobin of the notostracan branchiopod Lepidurus bilobatus has an apparent molecular weight of 680,000 and may exist in a dissociation-association equilibrium dependent on pH and ligand state. The pigment contains one heme per 18,000 g protein. However, attempts to dissociate the hemoglobin by harsh denaturing conditions results in a 33-34,000 molecular weight polypeptide chain as well as traces of some 62-64,000 molecular weight material. Limited proteolysis of this hemoglobin with subtilisin produces 14,800 and 16,500 dalton heme-containing polypeptides (domains) which bind oxygen reversibly. These domains, isolated by column chromatography, have a heme content similar to the intact pigment. It is proposed that the intact 34,000 dalton subunit of Lepidurus hemoglobin consists of two linearly linked oxygen binding domains. Oxygen binding properties of the intact hemoglobin show a low oxygen affinity with a slight Bohr effect. In contrast, the isolated domains display a relatively high oxygen affinity and lack a Bohr effect between pH 7.0 and 8.0. It is apparent that the intact 34,000 dalton polypeptide is necessary for the expression of the heterotropic interactions of the native pigment.     

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.     

Amino acid sequences of the aplpha and beta chains of adult hemoglobin of the tupai, Tupaia glis.

Globin prepared from hemoglobin of adult tupai (Tupaia glis) was separated into alpha and beta polypeptide chains by CM-cellulose column chromatography. The S-aminoethylated alpha polypeptide chain and S-carboxymethylated beta polypeptide chain were each digested with trypsin, and the sequences of all the peptides thus obtained were established. The ordering of these tryptic peptides in the alpha and beta polypeptide chains was deduced from the homology of their primary structures with that of human adult hemoglobin. In this way the primary structures of the alpha and beta polypeptide chains of tupai hemoglobin were established; 27 amino acids in the alpha polypeptide chain and 26 in the beta chain differ from those in human adult hemoglobin.     

Subunit structure of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus and amino acid sequence of the constituent polypeptide chain (IIC)

Tylorrhynchus cyanomethemoglobin reduced with dithiothreitol was separated by chromatofocusing into four heme-containing polypeptide chains (I, IIA, IIB, and IIC) and a non-heme chain (N). The molecular weights of chains IIA-C and N were confirmed to be the same by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on a 10-20% gradient gel. The molecular weight of chain IIC was determined to be 17,415 (including heme) from the amino acid sequence. Chain N constitutes less than 5% of the total protein and has the same NH2-terminal sequence, suggesting that it is derived from chain IIA during the isolation procedure. Tylorrhynchus hemoglobin consists of two types of subunit with molecular weights of 16,327 (chain I) and approximately 50,000, and the latter splits into chains IIA-C in the presence of a reducing agent. On the basis of the accurate value obtained for the molecular mass of chain IIC, it was concluded that the subunit of approximately 50,000 daltons is a trimer of heme-containing chains IIA, IIB, and IIC linked by disulfide bonds. The cysteine residue at position 5 and the arginine at position 10 are conserved in the four heme-containing chains of Tylorrhynchus hemoglobin. The complete sequence of 149 residues of Tylorrhynchus chain IIC was determined. This sequence shows high homology with Tylorrhynchus chain I (Suzuki, T., Takagi, T., and Gotoh, T. (1982) Biochem. Biophys. Acta 708, 253-258) and Lumbricus chain AIII (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015).     

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of the cyclic 3',5'-AMP-binding protein from the muscle of the Nematode Ascaris suum

The cyclic 3',5'-AMP-binding protein was isolated from the muscle of Ascaris suum and purified to apparent homogeneity. It migrated as a protein with a relative Mr 54,000 on electrophoresis under denaturing conditions. On gel filtration columns it was eluted at a volume corresponding to a protein of Mr greater than 200,000 under conditions which kept the cyclic 3',5'-AMP-binding property intact. The purified catalytic subunit of protein kinase from Ascaris and the C subunit of cyclic 3',5'-AMP-dependent protein kinase from bovine heart were inhibited by the cyclic 3',5'-AMP-binding protein. Gel filtration studies indicated the formation of a stable protein complex between the protein kinase and the cyclic 3',5'-AMP-binding protein from Ascaris.     

Primary structure of two linker chains of the extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus

Two types of linker subunits (linkers 1 and 2) of the extracellular hemoglobin of Tylorrhynchus heterochaetus have been isolated as disulfide-linked homodimers by C18 reverse-phase chromatography. These subunits constituted 6 and 13%, respectively, of total protein area on the chromatogram. The complete amino acid sequences of linkers 1 and 2 were determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase, trypsin, chymotrypsin, Staphylococcus aureus V8 protease, pepsin, and endoproteinase Asp-N. The linker 1 consisted of 253 amino acid residues (the calculated molecular mass, 28,200 Da), while the linker 2 consisted of 236 residues (26,316 Da). The two chains showed 27% sequence identity. The amino acid sequences of Tylorrhynchus linkers 1 and 2 also showed 23-27% homology with the recently determined sequence of a linker chain of Lamellibrachia hemoglobin (Suzuki, T., Takagi, T., and Ohta, S. (1990) J. Biol. Chem. 265, 1551-1555). In the three linker chains, half-cystine residues were highly conserved; 8 out of 13 residues are identical, suggesting that such residues would contribute to the formation of intrachain disulfide bonds essential for the protein folding of the linker polypeptides. Based on the exact molecular masses of the linker and the heme-containing subunits, the molar ratios estimated for the subunits and the minimum molecular weights per 1 mol of heme, a model is proposed for the subunit structure of the Tylorrhynchus hemoglobin, consisting of 216 polypeptide chains, 192 heme-containing chains, and 24 linker chains.     

Isolation of polypeptide chains with heme from the extracellular hemoglobin of Amphitrite ornata (Polychaeta, Annelida)

From the extracellular hemoglobin of Amphitrite ornata four constituent polypeptide chains containing heme and designated AI, AII, BI and BII according to the elution order were obtained by DE52-cellulose ion-exchange chromatography with dithiothreitol (DTT) as a reducing reagent. The NH2-terminal sequences for the chains are AI, Asp-Ser-Asn-Ala; AII, Glu-Tyr-Thr; BI, Asp-Phe-Asn-Thr; and BII, Asp-Ser-Glu. Each of the isolated chains showed spectra similar to those of vertebrate hemoglobins, and they bound oxygen reversibly. Acid urea polyacrylamide gel electrophoresis separated four bands, corresponding to the isolated chain, from the intact extracellular hemoglobin reduced with DTT. These results and our failure to detect an appreciable amount of non-heme protein suggest that the extracellular hemoglobin of A. ornata is composed of four polypeptide chains, each containing a heme.     

Linked functions in allosteric proteins: Extension of the concerted (MWC) model for ligand-linked subunit assembly and its application to human hemoglobins

The allosteric model of Monod et al. (1965) (MWC) has been extended to take into account the effects of subunit dissociation. The problem is formulated theoretically in terms of a general model for two allosteric species (dimers and tetramers) linked by a polymerization reaction. Relationships are presented for interpreting the dimer-tetramer association constants in terms of allosteric model parameters.Sub-cases of the general model were tested against recent experimental data on the oxygenation-linked dimer-tetramer equilibria in normal human hemoglobin and in the variant hemoglobin Kansas (β₁₀₂, Asp → Thr). The objectives of these analyses were: (1) to find the simplest models capable of describing the linked dimer-tetramer equilibria in the two hemoglobin systems, and (2) to evaluate the corresponding model parameters so that allosteric properties of the two hemoglobins may be compared.In the simplest version of the model, the dimer is half of an R-state tetramer. This model was found to be excluded unequivocally by the data for both normal hemoglobin and hemoglobin Kansas when the α and β chains have equal binding affinities. When this two-state model was modified to permit non-equivalent affinities for the chains, the model could be fitted to hemoglobin Kansas, but not to hemoglobin A. A model, in which the dimers are allowed to exist in a state different from the tetramer R state, was found to be consistent with the data for hemoglobin A, with equivalent binding by the α and β chains. For hemoglobin A, the unliganded R-state tetramers have a different subunit dissociation energy from that of fully liganded R-state tetramers. The simplest model capable of describing both hemoglobin A and hemoglobin Kansas was obtained by extending this three-state model to permit (but not require) functional non-equivalence of the α and β chains. For these MWC models, unique estimates were obtained for the model parameters.The allosteric constants for tetrameric hemoglobins A and Kansas are approximately equal. The value obtained from hemoglobin A is similar to previous estimates, whereas the value for hemoglobin Kansas is lower than previously estimated (Edelstein, 1971) by approximately two orders of magnitude. The low affinity of hemoglobin Kansas tetramer does not arise from an unusually high allosteric constant favoring the T-state species. It is largely the consequence of a greatly reduced oxygen affinity of β chains in the T state, and reduced values for the ratio between affinities in the R and T states.     

Primary structure of hemoglobin from cobraNaja naja naja

Cobra snakeNaja naja naja hemoglobin shows four bands on Triton electrophoresis. We present the primary structure of oneα and oneβ chain. The separation of polypeptide chains was achieved by ion exchange chromatography on carboxymethyl cellulose column. The amino acid sequence was established by automatic Edman degradation of the native chains and tryptic and hydrolytic peptides in a gas-phase sequencer. The structural data are compared with those of human and other reptile hemoglobins and reveal not only large variations from human but within reptiles. The amino acid exchanges involve several subunit contacts and heme binding sites. This is the first study on the hemoglobin of a land snake. There are only two amino acid sequences of sea snake hemoglobin (Microcephalophis gracilis gracilis andLiophis miliaris) reported in the literature.