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C Fronticelli

Publications and source records attributed to C Fronticelli.

At least 55 records · Page 3Linked to original sources
PubMed1989

Local phenomena and distribution of molecular species during the unfolding of heme-free myoglobin in the presence of GdnHCl and urea as seen by time-resolved fluorescence spectroscopy.

We have used time-resolved fluorescence spectroscopy for following the unfolding of apomyoglobin in urea and guanidine hydrochloride (GdnHCl). The data have been compared with those obtained using classical techniques such as CD and steady-state emission spectroscopy. Both the average intensity of the lifetimes and the size of the librational cone of the fluorophores, as measured by time-resolved fluorescence, increased with denaturant concentration and their changes largely preceded the modifications detectable with CD and the shift of the maximum of emission spectra. The data indicate that the changes in the local environments of the tryptophans were completed when the global modification monitored by CD and the emission spectra was still minimal. This suggests that an initial event in the denaturation of apomyoglobin is localized at the tryptophan residues. The correlation times of native apomyoglobin showed the rotational diffusion characteristics of a rigid rotor. In 3.6 M GdnHCl and 7.5 M urea, where the secondary structure is practically absent, the correlation times of the two systems became very short, as expected from the motion of a flexible polymer. In GdnHCl, under conditions of partial unfolding, it was not possible to detect the presence of native totally folded molecular species.

Apoproteins↗
PubMed1988

Modulation of oxygen affinity in hemoglobin by solvent components. Interaction of bovine hemoglobin with 2,3-diphosphoglycerate and monatomic anions.

In the absence of Cl- in Hepes buffer at pH 7.4, the oxygen affinity of bovine and human hemoglobin is equally sensitive to 2,3-diphosphoglyceric acid. The low oxygen affinity measured for bovine hemoglobin at physiological salt concentration can be explained by the high affinity of Cl- anions for oxygen-linked sites that are absent in human hemoglobin. Bovine hemoglobin can discriminate between the different halogens in the sense that different halide concentrations are necessary to produce the same P50. Competition experiments indicate that the halogens interact with the same oxygen-linked sites. In agreement with the different affinities for halides, the Bohr effect of bovine hemoglobin is larger in the presence of Cl- than in that of Br- and there is good agreement between the number of protons and anions exchanged with the solvent upon oxygenation of bovine hemoglobin.

Animals↗
PubMed1988

Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry.

We used 2 GHz harmonic content frequency-domain fluorescence to measure the intensity and the anisotropy decays from the intrinsic tryptophan fluorescence from human hemoglobin (Hb). The tryptophan intensity decays are dominated by a short-lived component which accounts for 35-60% of the total steady state intensity. The decay time of this short component varies from 9 to 27 ps and this component is sensitive to the ligation state of Hb. Our error analyses indicate the uncertainty is about +/- 3 ps. The intensity decays also show two longer lived components near 0.7 and 8 ns, which are probably due either to impurities or to Hb molecules in conformations which do not permit energy transfer. The anisotropy decays indicate the tryptophan residues in Hb are highly mobile, with apparent correlation times near 55 ps.

Carboxyhemoglobin↗
PubMed1988

Time-resolved emission spectra of hemoglobin on the picosecond time scale.

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.

Hemoglobins↗
PubMed1988

Bovine hemoglobin as a basis for artificial oxygen carriers.

Under physiological conditions of pH and [Cl-] ions the oxygen affinity of bovine hemoglobin is lower than that of human hemoglobin. The difference tends to disappear at low ionic strength, while it increases at high ionic strength. In the presence of Cl- ions bovine hemoglobin is not sensitive to 2,3-DPG, while in the absence of Cl- ions human and bovine hemoglobin respond to 2,3-DPG in a similar way. This is due to a high preferential binding of halogens by the deoxy-conformation in the bovine system. Reaction of deoxy-bovine hemoglobin with 2,3-dibromo-salycyl-fumarate results in a decreased oxygen affinity. Compounds can be purified by anion exchange chromatography which have the sedimentation velocity of tetrameric hemoglobins. They fail to dissociate into dimers at acid pH because of the presence of intramolecular crosslinks. Reverse phase chromatography shows that both kind of subunits are modified by the reaction. The half time of retention in rats of these tetramers is near 5 h. Bovine red cells do not contain 2,3-DPG, therefore they can be stored in the cold in saline for at least 2 months without significant modifications of their oxygen affinity. The oxygen affinity of bovine red cells can be modulated by addition and subtraction of Cl- ions from and by changing the pH of the media.

2,3-Diphosphoglycerate↗
PubMed1986

Librational motions of membrane-embedded Ca2+-ATPase of sarcoplasmic reticulum labeled with fluorescein isothiocyanate.

Continuing our investigation of the relationships between internal motions and functional properties of soluble and membrane-bound proteins we have explored the lifetimes and correlation times associated with the fluorescence emission of fluorescein-labeled Ca2+-dependent ATPase of sarcoplasmic reticulum. The emission was characterized by two lifetime components near 1.8 and 4.1 ns, probably due to exposure of the probe to environments of different polarities. The time-dependent anisotropy showed the presence of two correlation times near 0.8 and 6.6 ns. The shorter correlation time was due to motions of the probe around its point of attachment on the surface of the protein. The longer correlation time indicated the presence of internal motions of the protein. Both lifetimes and correlation times were insensitive to temperature between 2 and 10 degrees C. They were also insensitive to addition and removal of 100 microM free Ca2+.

Animals↗
PubMed1986

Bovine hemoglobin as a potential source of hemoglobin-based oxygen carriers: crosslinking with bis(2,3-dibromosalycyl)fumarate.

Reaction in anaerobic conditions of bovine hemoglobin with bis(2,3-dibromosalycyl)fumarate resulted in new derivatives with P50 in excess of 40 mmHg, as determined at 37 degrees C in 0.15 M Cl- at pH 7.4. Although the chromatographic preparations indicated some heterogeneity of the reacted material, the proteins obtained were homogeneous with regard to sedimentation velocity, which showed the presence of only nondissociable tetrameric species. SDS gel electrophoresis showed the presence of a new band with a mobility corresponding approximately to a molecular mass of 32 kDa, indicating the presence of covalent intramolecular crosslinks between subunit pairs. Chromatographic analyses indicated that both alpha and beta chains were chemically modified. The retention times in rats of the crosslinked hemoglobin was 10-times longer than that of untreated hemoglobin.

Animals↗
PubMed1986

Gelling properties of apohemoglobin S alone and in mixtures with hemoglobin S.

Apohemoglobin S formed a gel in the cold (5 degrees C) with a protein concentration in the supernatants after centrifugation of the gels (Csat) near 27 g/dl, in 0.02 M phosphate buffer at pH 7.2. Under the same experimental conditions in mixtures of apohemoglobin S and deoxyhemoglobin S the solubility of hemoglobin S in the cold was decreased from Csat greater than 40 g/dl in the absence to about 18 g/dl in the presence of apohemoglobin S. Conversely, in the same mixture, Csat of apohemoglobin S was decreased to about 5 g/dl. Also, gelling occurred in mixtures of oxyhemoglobin S and its apoderivative. Apohemoglobin A alone did not form gels; however, it induced fiber formation in deoxyhemoglobin S in the cold; unlike apohemoglobin S, it was not included in the precipitate. Gels of apohemoglobin S were not birefringent, and inspection at the electron microscope failed to show the presence of organized structures. Excluded volume effects were probably at the origin of the decreased solubility of hemoglobin S and apohemoglobin S in the presence of each other.

Apoproteins↗
PubMed1986

Conformational properties of membrane-bound fumarate reductase of Escherichia coli.

Anaerobically grown cells of Escherichia coli harboring the plasmid pFRD63 over-produce fumarate reductase, a membrane-bound complex localized in the inner membrane of the cell, where this enzyme represents at least 90% of the total membrane proteins (B. D. Lemire, J. J. Robinson, and J. H. Weiner (1982) J. Bacteriol. 152, 1126-1131). Preparations of inner membrane fractions suspended in 40% sucrose are optically clear, allowing optical spectroscopic measurements. Circular dichroism spectra showed that between pH 6 and 11 the secondary structure of the enzyme is at least 55% in alpha helix and that above pH 11 the structure abruptly changes to a beta-like conformation. The same phenomenon is observed in samples solubilized in the nonionic detergent C12E9. Absorption spectra of the enzyme either membrane bound or solubilized in detergents or exposed to alkaline pH showed that the accessibility of the active site to solvent components is modulated by the interaction of the protein with the membrane. Solubilization of the membrane-bound enzyme with 1% Triton X-100 or C12E9 produced a decrease in ellipticity and in enzymatic activity.

Cell Membrane↗
PubMed1985

Anion Bohr effect of human hemoglobin.

The pH dependence of oxygen affinity of hemoglobin (Bohr effect) is due to ligand-linked pK shifts of ionizable groups. Attempt to identify these groups has produced controversial data and interpretations. In a further attempt to clarify the situation, we noticed that hemoglobin alkylated in its liganded form lost the Bohr effect while hemoglobin alkylated in its unliganded form showed the presence of a practically unmodified Bohr effect. In spite of this difference, analyses of the extent of alkylation of the two compounds failed to identify the presence of specific preferential alkylations. In particular, the alpha 1 valines and beta 146 histidines appeared to be alkylated to the same extent in the two proteins. Focusing our attention on the effect of the anions on the functional properties of hemoglobin, we measured the Bohr effect of untreated hemoglobin in buffers made with HEPES [N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid], MES [2-(N-morpholino)ethanesulfonic acid], and MOPS [3-(N-morpholino)propanesulfonic acid], which being zwitterions do not need addition of chlorides or other anions for reaching the desired pH. The shape acquired by the Bohr effect curves, either as pH dependence of oxygen affinity or as pH dependence of protons exchanged with the solution, was irreconcilable with that of the Bohr effect curves in usual buffers. This indicated the relevance of solvent components in determining the functional properties of hemoglobin. A new thermodynamic model is proposed for the Bohr effect that includes the interaction of hemoglobin with solvent components. The classic proton Bohr effect is a special case of the new theory.

Acetates↗
PubMed1985

Circular dichroism kinetics of acid denaturation of hemoglobin and of its beta-subunits.

Human hemoglobin and its isolated beta-subunits were denatured by addition of HCl so as to reach final pH values ranging from 2.0 to 3.2. The beta-subunits were alkylated in both the beta 93 and beta 112 cysteines; this treatment makes the beta-subunits monomeric. The kinetics of acid denaturation of the two proteins was followed spectropolarimetrically in the millisecond time range, measuring the changes in circular dichroism at 225 nm. At all pH values, in both systems, the decay of ellipticity could be simulated by two exponentials. The initial ellipticity values of the solutions, obtained by extrapolation at zero time, were those expected for the native proteins. The rates of denaturation were lower in the hemoglobin system than in the isolated monomeric beta-subunits. The data suggest that in the tertiary structure of hemoglobin and beta IAA there are different domains which unfold at different rates upon exposure to acid.

Circular Dichroism↗
PubMed1984

Solvent regulation of oxygen affinity in hemoglobin. Sensitivity of bovine hemoglobin to chloride ions.

Under physiological conditions of pH (7.4) and chloride concentration (0.15 M), the oxygen affinity of bovine hemoglobin is substantially lower than that of human hemoglobin. Also, the Bohr effect is much more pronounced in bovine hemoglobin. Numerical simulations indicate that both phenomena can be explained by a larger preferential binding of chloride ions to deoxyhemoglobin in the bovine system. Also, they show that the larger preferential binding may be produced by a decreased affinity of the anions for oxyhemoglobin, thereby stressing the potential relevance of the oxy conformation in regulating the functional properties of the protein. The conformation of the amino-terminal end of the beta subunits appears to regulate the interaction of hemoglobin with solvent components. The pronounced sensitivity of the oxygen affinity of bovine hemoglobin to chloride concentration and to pH suggests that in bovine species these are the modulators of oxygen transport in vivo.

Animals↗
PubMed1984

Specialized functional domains in hemoglobin: dimensions in solution of the apohemoglobin dimer labeled with fluorescein iodoacetamide.

The fluorescence characteristics of 8-anilino-naphthalene-1-sulfonic acid (ANS) coupled to apohemoglobin and to apohemoglobin labeled with fluorescein iodoacetamide (FIA) at beta-93 have been compared. The quenching of emission of ANS produced by FIA was measured both with steady-state and with time-resolved techniques. In this system the emission of ANS in the beta-heme pockets was totally quenched by FIA at beta-93. Steady-state measurements indicated a 57% efficiency of energy transfer between ANS in the alpha-heme pockets and FIA at beta-93. Time resolution showed that the initial (unquenched) lifetime of ANS was 18.2 ns. In the presence of FIA two new components were generated with lifetimes of 2.0 and 6.6 ns. Assuming a random orientation of the probes, the distances inferred from these measurements were near 4.6 and 3.6 nm for the time-resolved and near 48 A for the steady-state measurements. In the tridimensional model of hemoglobin the distance between the iron atom of the alpha 1 chains and the SH group of the beta 1 chains at position 93 is 3.6 nm in oxyhemoglobin and 4.1 nm in deoxyhemoglobin. To these distances 0.5-1.0 nm may be added to allow for the dimensions of the probes. Thus it appears that removal of the heme fails to produce any important enlargement of the molecule. On the contrary, the data suggest a slight shrinking of apohemoglobin, which may be consistent with a collapse of the heme pocket when heme is removed. The rest of the molecule does not seem to be greatly affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1984

Solvent perturbation evidence for a two-state system regulated by calcium in sarcoplasmic reticulum ATPase.

Perturbation of sarcoplasmic reticulum ATPase with the nonionic detergent C12E8 is modulated by the amount of free Ca2+ present in the solvent prior to the addition of detergent. CD measurements show that the enzyme exists in solution in two different conformations that react differently with the detergent. They probably represent the free enzyme, and its complex with Ca2+. On this assumption, titrations with increasing amounts of Ca2+ produced data superimposable on curves obtained measuring Ca2+ bound to sarcoplasmic reticulum vesicles.

Animals↗
PubMed1983

Neohemoglobins as blood substitutes.

The brackets in fig 5, show a cluster of cross-linked human and bovine hemoglobins whose oxygen affinity is lower than that of untreated human SFH. Also, they have a stabilized tetrameric structure, and the combination of these characteristics makes these compounds very promising as oxygen carriers in cell-free blood substitutes.

Animals↗
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↗
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↗