Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104.Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.     

cDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.     

A comparison of the carbohydrate binding properties of twoDolichos biflorus lectins

The carbohydrate binding properties of theDolichos biflorus seed lectin and DB58, a vegetative tissue lectin from this plant, were compared using two types of solid phase assays. Both lectins bind to hog blood group A + H substance covalently coupled to Sepharose 4B and this binding can be inhibited with free blood group A + H substance. However, the binding of the seed lectin is inhibited byD-GalNAc whereas DB58 binding was not inhbited by any monosaccharide tested, thus suggesting that its carbohydrate combining site may be more extensive than that of the seed lectin. The activities of these two lectins also differ from one another in ability to recognize blood group A + H substance adsorbed on to plastic and in the effects of salt and urea on their carbohydrate binding activities. Neither lectin showed glycosidase activity with p-nitrophenyl -D-GalNAc or p-nitrophenyl -D-GalNAc.     

Weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume Dolichos biflorus

The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation, which becomes important when they interact with multivalent glycoconjugates, for instance those on cell surfaces. Recently, it has become clear that certain lectins form weakly associated oligomers. This phenomenon may play a role in the regulation of receptor crosslinking and subsequent signal transduction. The crystal structure of DB58, a dimeric lectin from the legume Dolichos biflorus reveals a separate dimer of a previously unobserved type, in addition to a tetramer consisting of two such dimers. This tetramer resembles that formed by DBL, the seed lectin from the same plant. A single amino acid substitution in DB58 affects the conformation and flexibility of a loop in the canonical dimer interface. This disrupts the formation of a stable DBL-like tetramer in solution, but does not prohibit its formation in suitable conditions, which greatly increases the possibilities for the cross-linking of multivalent ligands. The non-canonical DB58 dimer has a buried symmetrical alpha helix, which can be present in the crystal in either of two antiparallel orientations. Two existing structures and datasets for lectins with similar quaternary structures were reconsidered. A central alpha helix could be observed in the soybean lectin, but not in the leucoagglutinating lectin from Phaseolus vulgaris. The relative position and orientation of the carbohydrate-binding sites in the DB58 dimer may affect its ability to crosslink mulitivalent ligands, compared to the other legume lectin dimers.     

Photoaffinity labeling of the adenine binding site of the lectins from lima bean, Phaseolus lunatus, and the kidney bean, Phaseolus vulgaris

8-Azidoadenine was employed as a photoaffinity probe of the adenine binding site of the seed lectin from lima beans and from Phaseolus vulgaris erythroagglutinin. This compound was shown to (a) bind competitively to the adenine binding site of these lectins and (b) exhibit enhanced binding in the presence of 1,8-anilinonaphthalenesulfonic acid in the same manner as adenine. The presence or absence of 1,8-anilinonaphthalenesulfonic acid during labeling caused no change in the peptide maps of either lectin when digested with trypsin. The peptide maps of each lectin showed one major peak of radioactivity. Sequencing of the corresponding tryptic peptide from lima bean lectin indicated the primary structure to be Val-Leu-Ile-Thr-Tyr-Asp-Ser-Ser-Thr-Lys. The sequence of the labeled peptide isolated from P. vulgaris erythroagglutinin was Thr-Thr-Thr-Trp-Asp-Phe-Val-Gly-Glu-Asn-Glu-Val-Leu-Ile-Thr-Tyr, which corresponded to residues 173-190 of the cDNA-derived sequence (Hoffman, L. M., and Donaldson, D. D. (1985) EMBO J. 4, 883-889). Residues 186-190 (italicized) are identical to the first five amino acids in the lima bean lectin peptide. The peptides are located at the COOH-terminal half of the lectin and show extensive homology with other legume lectins.     

Tyrosine-371 contributes to the positive cooperativity between the two cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural changes that are induced as a consequence of cAMP-binding. Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 [Bubis, J., & Taylor, S.S. (1987) Biochemistry 26, 3478-3486]. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tyrosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes. A single base change converted Tyr-371 to Phe. This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer. The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent Kd(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99. The Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.     

Flow injection analysis of binding reaction between fluorescent lectin and cells

A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y. Oda, M. Kinoshita, and K. Kakehi, Anal. Biochem. 254, 41-48, 1997). However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1. 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h.     

ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue

The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal structure of the carbohydrate-recognition domain of the glycoprotein sorting receptor p58/ERGIC-53 reveals an unpredicted metal-binding site and conformational changes associated with calcium ion binding

p58/ERGIC-53 is a calcium-dependent animal lectin that acts as a cargo receptor, binding to a set of glycoproteins in the endoplasmic reticulum (ER) and transporting them to the Golgi complex. It is similar in structure to calcium-dependent leguminous lectins. We have determined the structure of the carbohydrate-recognition domain of p58/ERGIC-53 in its calcium-bound form. The structure reveals localized but large conformational changes in relation to the previously determined metal ion-free structure, mapping mostly to the ligand-binding site. It reveals the presence of two calcium ion-binding sites located 6A apart, one of which has no equivalent in the plant lectins. The second metal ion-binding site present in that class of lectins, binding Mn(2+), is absent from p58/ERGIC-53. The absence of a short loop in the ligand-binding site in this protein suggests that it has adapted to optimally bind the high-mannose Man(8)(GlcNAc)(2) glycan common to glycoproteins at the ER exit stage.     

Structural characterization of a Vatairea macrocarpa lectin in complex with a tumor-associated antigen: A new tool for cancer research

Legume lectins are the most thoroughly studied group of lectins and have been widely linked to many pathological processes. Their use as immunohistochemistry markers for cell profiling and cancer diagnosis have made these molecules important tools for immunological studies and have stimulated the prospection and characterization of new lectins. The crystal structures of a recombinant seed lectin from Vatairea macrocarpa (rVML) and its complexes with GalNAcα1-O-Ser, GalNAc and α-lactose, have been determined at 1.90, 1.97, 2.70 and 1.83 Å resolution, respectively. Small angle X-ray scattering and calorimetry assays have confirmed the same pH stable oligomerization pattern and binding profiles proposed for its wild-type counterpart. In silico analyzes have explored the potential of this recombinant lectin as new tool for cancer research through a comparative profile with other legume lectins widely used for cancer diagnosis and prognosis. The results suggest the recognition of specific epitopes exhibited on different cancer cells as a process that relies on the disposition of hydrophobic clusters and charged regions around the lectin carbohydrate-binding site, favouring the anchorage of different groups in the antigen boundaries, highlighting the different potential of each analyzed lectin. In conclusion, the experimental results and comparative analysis show that rVML is as a promising tool for cancer research, able to bind with high affinity specific tumor-associated antigens, highly stable and easily produced.     

Purification and properties of a lectin from lonchocarpus capassa (apple-leaf) seed

A lectin has been purified from L. capassa seed by ammonium sulphate fractionation and affinity chromatography on a column of D-galactose-derivatized Sepharose. The lectin is a glycoprotein which contains 3.8% neutral carbohydrates comprised of mannose, N-acetylglucosamine, xylose and fucose. The subunit M, of the lectin is 29 000, it has only alanine as N-terminal amino acid and contains 240 amino acids with a high content of acidic and hydroxy amino acids, single residues of methionine and histidine and the absence ofcystine. The lectin of L. capassa seed is a metalloprotein in that it contains 0.8 mol Ca²⁺ and 0.4 mol Mn²⁺ per mol. It agglutinates untreated human A, O and B type erythrocytes and rabbit erythrocytes. N-Acetyl-D-galactosamine was the best inhibitor. D-Galactose and various carbohydrates containing this sugar inhibit the hemagglutinating activity of the lectin. The lectin is also inhibited by D-glucose. The amino-terminal sequence of the lectin from L. capassa seed shows a significant degree of homology with many lectins from leguminous plants and is related to concanavalin A by a circularly permuted sequence homology.     

Binding of isofraxidin to bovine serum albumin

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5 x 10(-6) mol x L(-1) and drug concentration in the range of 1.67 x 10(-6) mol x L(-1) to 2.0 x 10(-5) mol x L(-1). Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug-binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -17.63 kJ x mol(-1) and 51.38 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA.     

Fractionation of lymphocytes using affinity chromatography with 9 lectins

Lymphocyte subclasses from normal peripheral blood have been fractionated by affinity chromatography with lectins. Concanavalin A (Con A), Lens culinaris lectin (LC), Pisum sativum lectin (PS), Phaseolus vulgaris lectin (PHA), Dolichos biflours lectin (DB), Glicine max lectin (SBA), Ricinus communis lectin (RCA II), Tetragonolobus purpureus lectin (TP) and Triticum vulgaris lectin (WGA), were coupled to Sepharose 6MB, and lymphocytes labelled with 125I were eluted through the chromatographic columns. The binding of lymphocytes to WGA and SBA lectins was 32% and 13% respectively. The binding to the other lectins tested were found to be between 32% and 13%. When solutions of increasing concentrations of specific sugar were added to the columns a progressive elution of bound lymphocytes was observed. These results indicate the existence of a large range of lymphocyte subclasses, with different binding capacity to lectins, which was a function of the receptor number or/and receptor affinity to each lectin. Furthermore, these two parameters were found to vary in each functional population. Even though all the lymphocytes had lectin receptors, T lymphocytes showed higher affinity for Con A, PHA and TP lectins, while B lymphocytes appeared to be more specific for LC, PS, SBA, DB, RCAII and WGA lectins.     

Properties of the lectin from the hog peanut (Amphicarpaea bracteata)

An N-acetyl-D-galactosamine-specific lectin has been isolated from the two seed forms of the hog peanut (Amphicarpaea bracteata) using an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1,3)-[alpha-L-Fuc-(1,2)]-beta-D-Gal (Synsorb A). The affinity-purified lectin appears to be identical in both seed types. Gel filtration on Sephadex G-200 gives a single symmetrical peak corresponding to Mr 135,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows four subunit forms, each of which contains carbohydrate. Limited amino terminal sequencing indicates heterogeneity in two of the first 10 residues. The lectin contains no cysteine. There are four equivalent, noninteracting GalNAc binding sites per 135,000-Da molecule, having an association constant for methyl N-acetyl-alpha-D-galactosaminide of 4.0 X 10(4) M-1. Precipitin and hapten inhibition studies show the lectin to be specific for terminal, nonreducing D-GalNAc units, with a preference for the alpha-anomer and enhanced specificity for the disaccharide, GalNAc alpha 1,3GalNAc. There is also a single adenine binding site per Mr 135,000 lectin molecule with an association constant of 1.3 X 10(6) M-1.     

Differences in properties of peanut seed lectin and purified galactose- and mannose-binding lectins from nodules of peanut

Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal galactose - Gle glucose - GNL galactose-binding nodule lectin - Fru fructose - MNL mannosebinding nodule lectin - M _r rerative molecular mass - PBS phosphate-buffered saline - PSL peanut seed lectin - SDS sodium dodecyl sulphate - Sorb sorbitol     

Interaction with lectins and differential wheat germ agglutinin binding of pyocin 103-sensitive and -resistant Neisseria gonorrhoeae

Strains of Neisseria gonorrhoeae were treated with pyocin 611 131 (pyocin 103) from Pseudomonas aeruginosa PA103, and isogenic resistant variants were isolated. The interaction of pyocin-sensitive and isogenic pyocin-resistant strains with wheat germ agglutinin (WGA) agglutinated all pyocin-sensitive, but not pyocin-resistant, strains. Binding of WGA to three pyocin-sensitive strains and their isogenic pyocin-resistant variants was examined quantitatively by using fluorescein-conjugated lectin. Pyocin-resistant strains maximally bound one-third to one-eighth the quantity of WGA bound by isogenic-sensitive strains. Linear Scatchard plots revealed homogeneous WGA-binding sites on three pyocin-sensitive and one pyocin-resistant strains. Biphasic Scatchard plots, obtained with two pyocin-resistant strains, show that WGA-binding sites in these strains are heterogeneous. The number of WGA-binding sites for pyocin-sensitive organisms ranged from 8 x 10(5) to 1 x 10(6) sites per coccus and from 1 x 10(5) to 3 x 10(5) sites per coccus for pyocin-resistant strains. The apparent association constant for WGA binding to pyocin-sensitive strains ranged from 3 x 10(6) to 6 x 10(6) liters/mol and from 6 x 10(6) to 1 x 10(7) liters/mol for pyocin-resistant strains. Gonococcal lipopolysaccharide was shown to serve as the pyocin 103 receptor by inhibition of pyocin activity. Lipopolysaccharide from a pyocin 103-resistant strain was not able to inhibit pyocin 103 activity. Pyocin 103 resistance was correlated with a structural alteration involving N-acetylglucosamine residues in gonococcal lipopolysaccharide. Based on interactions with wheat germ, soybean, and ricin lectins, a model of lipopolysaccharide structure in N. gonorrhoeae is presented.     

Fluorescence study of steroid hormone binding activity of Helix pomatia agglutinin

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin, found in the reproductive gland of a Roman snail. The present study has shown that HPA, in addition to its carbohydrate binding capacity possesses a hydrophobic binding activity. This protein binds with high affinity (k(D)=1.9-2.4 microM) steroid hormones: testosterone and progesterone, identified as putative ligands for the animal lectin HPA. Additionally, we have found that this lectin also interacts with adenine (k(D)=5.4+/-0.5 microM) and arylaminonaphthalene sulfonate TNS (k(D)=12+/-0.3 microM). Binding of HPA to hormones and adenine was accompanied by a significant increase of the intrinsic Trp fluorescence (up to 50%), characterizing the conformational changes in the lectin molecule. The hyperbolic shape of the binding curves indicated one high affinity site for the two steroid hormones and adenine, and more than one hydrophobic site for TNS, showed by the sigmoidal curve fit and Hill coefficient of (n(H)=1.5+/-0.2). Hormones and adenine compete for an identical binding site, suggested to occupy the central hydrophobic cavity of the HPA hexamer. Fluorescence resonance energy transfer (FRET) was applied to calculate the intramolecular distance between TNS and Trp chromophores.     

The mutation K30D disrupts the only salt bridge at the subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis and changes the mechanism of cooperativity.

The subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis, HbI, is stabilized by a network of interactions that involve several hydrogen-bonded structural water molecules, a hydrophobic patch, and a single, symmetrical salt bridge between residues Lys-30 and Asp-89. Upon mutation of Lys-30 to Asp, the interface is destabilized markedly. Sedimentation equilibrium and velocity experiments allowed the estimate of the dimerization constants for the unliganded (K(1,2D) = 8 x 10(4) M(-1)) and for the CO-bound (K(1,2L) = 1 x 10(3) m(-1)) and oxygenated (K(1,2L) = 70 m(-1)) derivatives. For the oxygenated derivative, the destabilization of the subunit interface with respect to native HbI corresponds to about 8 kcal/mol, an unexpectedly high figure. In the K30D mutant, at variance with the native protein, oxygen affinity and cooperativity are strongly dependent on protein concentration. At low protein concentrations (e.g. 1.2 x 10(-5) m heme), at which the monomeric species becomes significant also in the unliganded derivative, oxygen affinity increases and cooperativity decreases. At protein concentrations where both derivatives are dimeric (e.g. 3.3 x 10(-3) m heme), both cooperativity and oxygen affinity decrease. Taken together, the experimental data indicate that in the K30D mutant, the mechanism of cooperativity is drastically altered and is driven by a ligand-linked monomer-dimer equilibrium rather than being based on a direct heme-heme communication as in native HbI.