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E Bucci

Publications and source records attributed to E Bucci.

At least 163 records · Page 9Linked to original sources
PubMed1982

Folding domains as functional tools in allosteric systems: a heme-dependent domain in hemoglobin beta subunits.

We have investigated the denaturation by guanidine hydrochloride (Gdn X HCl), temperature, and pH of hemoglobin beta subunits and of the peptides beta (1-146), beta (56-146), and beta (1-55). The last peptide was insensitive to all of the three agents. In the other polypeptides denaturation by Gdn X HCl and temperature showed the presence of several structural domains characterized by different stabilities. Analyses of the data obtained in Gdn X HCl indicated the presence in beta subunits of an alpha-helical domain involving some 40 amino acids whose free energy of denaturation is only 2000 cal. This domain is heme dependent, and removal of heme in apo-beta (1-146) abolishes the presence of the domain as a structural entity. Acid denaturation reveals in beta subunits, apo-beta (1-146), and beta (56-146) the presence of buried histidines with very similar characteristics, indicating a similar tertiary structure in the three polypeptides. This suggests that removal of the heme produces an unfolding of beta subunits, involving preferentially the 1-55 portion of the chain. This portion of the polypeptide contributes substantially to the formation of the alpha 1 beta 2 interface in hemoglobin. The low stability of this domain implies a very small contribution to the stability of the system as a whole. Instead it makes it very sensitive to conformational attitudes of the heme, suggesting a role in the mechanism of ligand binding cooperativity and subunits interactions in hemoglobin.

Allosteric Site↗
PubMed1982

Librational modes in liganded and unliganded hemoglobin as seen by fluorescence spectroscopy.

Hemoglobin labeled with N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate at the beta-93 cysteine shows normal allosteric properties including the Bohr effect and heme-heme-interaction, and the oxygen affinity is somewhat increased. Viscosity-resolved correlation times obtained from direct measurement of the decay of fluorescence anisotropy show values of 4.15 +/- 0.23 and 9.13 +/- 0.46 ns for the liganded and unliganded derivatives, respectively, in 0.05 M tris buffer, pH 7.5 at 15 degrees C. Addition of 1 mM inositol hexaphosphate to the liganded and unliganded derivatives results in correlation times of 9.31 +/- 0.87 and 43.69 +/- 16.04 ns, respectively. Similar values of correlation times are obtained from Perrin plots. Considering that the correlation times expected for the single subunits, the dimers, and the tetramers of the system are approximately 10-15, 20-30, and above 40 ns, respectively, it can be concluded that the molecule of hemoglobin becomes increasingly rigid upon removal of ligands and addition of inositol hexaphosphate. These observations support the hypothesis of a functional relevance of the internal flexibility of hemoglobin.

Fluorescent Dyes↗
PubMed1982

The pK of the amino terminal groups of carbonmonoxy- and deoxyhemoglobin measured by dinitrophenylation in phosphate buffers.

The rate of reaction of the terminal valines of the alpha- and beta-chains of hemoglobin with 1-fluoro-2,4-dinitrobenzene was followed spectrophotometrically at 353 nm. The variation with pH of the rate of dinitrophenylation of these groups was measured for both carbonmonoxy- and deoxyhemoglobin. In carbonmonoxyhemoglobin the results indicated a pK near 6.7 and 7.7 for the amino terminal groups of the two kinds of subunits, and were attributed to the alpha- and beta-chains respectively. Removal of ligands produced an increase of 0.1 in both pK values and a decrease of 40% of the pH-independent kinetic constant for dinitrophenylation of the beta-subunits. These modifications are due to the conformational changes associated with ligand binding in the system. In phosphate buffers the contribution to the Bohr effect of the amino terminal residues of either chains is negligible.

Adult↗
PubMed1981

Molecular dynamics of hemoglobin subunits as seen by fluorescence spectroscopy.

Fluorescent conjugates of beta A subunits and their respective heme-free derivatives have been prepared in which a 1,5-N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonate probe has been specifically placed at the beta-93 or beta-112 cysteine. The fluorescence anisotropy decay and static fluorescence polarization of these conjugates have been examined. Fluorescence measurements have also been made using 1-anilino-8-naphthalenesulfonate complexes, as well as the intrinsic fluorescence of the tryptophan groups. For the cases of the beta-93 and beta-112 conjugates there is substantial evidence for internal rotational freedom of the subunits. The internal mobility of the polypeptide is especially pronounced for the beta-112 conjugate. In contrast, the 1-anilino-8-naphthalenesulfonate probe placed within the heme pocket shows no indication of any rotation, other than that associated with the entire beta-subunit. Tryptophan fluorescence has been measured for the apo-beta subunits and for the peptides beta (1-55) from hemoglobins A and S. Perrin-Weber plots show the presence of multiple rotational modes suggesting mobility of the tryptophan groups.

Anilino Naphthalenesulfonates↗
PubMed1980

Conformational changes in the hemoglobin S system as seen by proton binding.

The proton binding behavior of the carbon monoxy derivatives of hemoglobins A and S, the respective beta-subunits (in the native form and with the cysteines combined with p-mercuribenzoate), and the respective beta (1-55) peptides have been measured. The results show that in the systems obtained from hemoglobin S there is a group which shifts its pK from about 7.0 to 8.35 in the beta-subunits that were reacted with p-mercuribenzoate and to more than 9.0 in the beta (1-55) peptide. Proton nuclear magnetic resonance measurements indicate that in the peptide beta (1-55) from hemoglobin S this residue is the histidine at beta 2. It is proposed that this pK shift is due to the formation of a salt bridge between beta 2 His and beta 7 Glu. This structure would disrupt the first turn of the A helix of the beta s-subunits. Its stabilization by extramolecular contacts may be relevant to the mechanism of fiber formation of hemoglobin S.

Carboxyhemoglobin↗
PubMed1979

Conformation in solution of hemoglobin Osler (alpha 2 A beta 2 145 Tyr replaced by Asp).

Computer simulations of Gelin and Karplus ((1977) Proc. Natl. Acad. Sci. U.S.A. 74, 801-805) suggest that in hemoglobin upon ligation the penultimate tyrosyl residues of the subunits are not expelled from the hydrophobic pockets described in the crystals between the helices E and F (Perutz, M.F. (1970) Nature 228, 726-737). This implies that both the liganded and unliganded conformations of hemoglobin may be affected by mutations involving such residues. Investigation of the conformational behavior of liganded and unliganded hemoglobin Osler was conducted measuring the functional properties, the subunits dissociation, the CD and electronic spectra, the protons absorption upon interaction with polyanions, and the reactivity of the -SH groups of the protein. The results suggest that both the liganded and unliganded conformations of the system are affected by the mutation, confirming the anticipations of Gelin and Karplus on the relevance of tyrosine at beta 145 for both allosteric states of hemoglobin.

Alkylation↗
PubMed1979

Effect of polyanions on the kinetics of the reaction of apohemoglobin with carbonmonoxy heme.

The reaction of apohemoglobin with carbonmonoxy heme and with carbonmonoxy heme dimethyl ester was investigated in the presence and absence of inositol hexaphosphate. The binding stoichiometry of both heme derivatives to apohemoglobin was not affected by the presence of the polyphosphate, while, in both cases, the overall rate of recombination was substantially decreased. The absence of the negatively charged carboxyl groups in the dimethyl ester derivative of the heme indicated that the effect of inositol hexaphosphate on the reaction of apohemoglobin with heme was not due to electrostatic repulsions and resulted from conformational changes occurring upon the interaction of apohemoglobin with inositol hexaphosphate. Qualitative treatment of the kinetic data suggests that these conformational changes destabilize the intermediates of the reaction by increasing their redissociation into the original components. Also, benzenehexacarboxylate produced conformational changes in apohemoglobin and decreased its rate of reaction with carbonmonoxy heme, proving the aspecificity of the interaction of apohemoglobin with polyanions.

Apoproteins↗
PubMed1979

Interaction of human apohemoglobin with inositol hexaphosphate.

Experiments of sedimentation velocity and equilibrium indicate that in the presence of inositol hexaphosphate the degree of polymerization of apohemoglobin is shifted in favor of the formation of tetramers, with a maximum effect when the concentration of the polyphosphate is 1 mM. Above this concentration, a redissociation of the system into dimers is promoted. This phenomenon is probably due to the binding of inositol hexaphosphate to apohemoglobin with a stoichiometry higher than 1 mol of polyphosphate/4 subunits. The optical rotatory dispersion spectrum of apohemoglobin is also modified by its interaction with inositol hexaphosphate suggesting a small increase in the helical content of the protein. Measurements of circular dichroism in the near-UV region of the spectrum indicate that the environment of the aromatic chromophores of the protein such as tyrosine, phenyalanine, and tryptophan is not affected by the interaction. The presence of inositol hexaphosphate decreases the rate of reaction of the beta-93 cysteinyl residues of apohemoglobin with both p-mercuribenzoate and N-ethylmaleimide, suggesting a conformational change of the protein also at the level of its tertiary structure.

Apoproteins↗
PubMed1976

Conformational aspects of the interaction of polyanions with liganded beta chains of human hemoglobin.

The interaction of carbon monoxide beta chains with two allosteric effectors, namely inositol hexaphosphate and benzenehexacarboxylate, was studied. The sedimentation coefficient (s20,w) of the liganded beta chains was measured to be the same both in the presence and absence of the two effectors suggesting that the protein exists as a tetramer under the conditions of our titration and optical studies. The binding of benzenehexacarboxylate to the liganded beta chains was investigated by potentiometric titration in the pH range 6.7-8.0. The results at pH 7.4 showed a binding of 2 mol of benzenehexacarboxylate per tetramer, with an association constant of 1.26 X 10(4) 1. mol-1 at 20 degrees C. The Hill coefficient for the binding was determined to be 0.73. Similar experiments on the interaction of inositol hexaphosphate with the beta chains showed a binding of 2 mol of the effector per tetramer with identical Hill coefficient (0.737) and comparable association constants (0.88 X 10(4)1. mol-1). The value below unity of the Hill coefficient, found for the binding of the two effectors to the protein, probably reflected an anticooperativity produced by the different net electric charges of the free protein and the protein-effector complex. The difference in protons bound per mole of heme by the beta subunits in the presence and absence of benzenehexacarboxylate appeared consistent with the proposal that two groups per chain changed their pK from 6.6 to 7.4 upon the interaction. In the presence of benzenehexacarboxylate, the protonation of these groups appeared to be cooperative, suggesting a conformational change of the protein upon the binding. The absorption spectra of carbon monoxide beta chains in the Soret region was markedly altered by benzenehexacarboxylate and inositol hexaphosphate. The features in the difference spectra of the protein obtained with the two effectors were qualitatively identical and indicated changes in the heme environment produced by the interaction of the effectors with the beta chains. Concomitant changes in circular dichroism and optical rotatory dispersion of the liganded beta chains caused by the addition of the two effectors provided supporting evidence for the conformational change in the protein produced by the binding of the effectors.

Binding Sites↗
PubMed1975

Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.

The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G.D. Fasman ((1969), Biochemistry 8, 4108). In this way the helical content of the native and reconstituted alkylated beta subunits appeared to be near 76%, a value very near to that present in the same subunits in the hemoglobin crystal. The helical content of the apo-beta subunits in 0.04 M borate buffer at pH 9.6 decreased to a value near 45%. The helical content of the isolated peptides in electrolyte solutions was in any case near 10% indicating an almost complete loss of the structure that they have in the hemoglobin crystal. Cyanoheme reacted with the peptide beta(41-104), however, the reaction was not stoichiometric indicating a low affinity of the heme for the peptide. With the exception of the peptide beta(31-104), all of the other peptides recovered some of their helical structure when dissolved in 50% methanol. Notably also the apo-beta subunits did so suggesting that the loss of structure upon the removal of the heme could be in part due to the exposure of the heme pocket to water.

Amino Acids↗