Biomedical subjects

C Fronticelli

Publications and source records attributed to C Fronticelli.

At least 19 recordsLinked to original sources

PubMed2001

Heme pocket disorder in myoglobin: reversal by acid-induced soft refolding.

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.

Acids↗

PubMed2001

Molecular engineering of a polymer of tetrameric hemoglobins.

We have engineered a recombinant mutant human hemoglobin, Hb Prisca beta(S9C+C93A+C112G), which assembles in a polymeric form. The polymerization is obtained through the formation of intermolecular S-S bonds between cysteine residues introduced at position beta9, on the model of Hb Porto Alegre (beta9Ser --> Cys) (Bonaventura and Riggs, Science 1967;155:800-802). Cbeta93 and Cbeta112 were replaced in order to prevent formation of spurious S&bond;S bonds during the expression, assembly, and polymerization events. Dynamic light scattering measurements indicate that the final polymerization product is mainly formed by 6 to 8 tetrameric hemoglobin molecules. The sample polydispersity Q = 0.07 +/- 0.02, is similar to that of purified human hemoglobin (Q = 0.02 +/- 0.02), consistent with a good degree of homogeneity. In the presence of strong reducing agents, the polymer reverts to its tetrameric form. During the depolymerization process, a direct correlation is observed between the hydrodynamic radius and the light scattering of the system, which, in turn, is proportional to the mass of the protein. We interpret this to indicate that the hemoglobin molecules are tightly packed in the polymer with no empty spaces. The tight packing of the hemoglobin molecules suggests that the polymer has a globular shape and, thus, allows estimation of its radius. An illustration of an arrangement of a finite number of tetrameric hemoglobin molecules is presented. The conformational and functional characteristics of this polymer, such as heme pocket conformation, stability to denaturation, autoxidation rate, oxygen affinity, and cooperativity, remain similar to those of tetrameric human hemoglobin.

Circular Dichroism↗

PubMed2001

Allosteric free energy changes at the alpha 1 beta 2 interface of human hemoglobin probed by proton exchange of Trp beta 37.

The energetic changes that occur on ligand binding in human hemoglobin have been investigated by measurements of the exchange rates of the indole proton of Trpbeta37(C3). The Trpbeta37 residues are located in helices C of the beta-subunits and are involved in contacts with the segments FG of the alpha-subunits at the interdimeric alpha1beta2 and alpha2beta1 interfaces of the hemoglobin tetramer. In the quaternary structure change that accompanies ligand binding to hemoglobin, these contacts undergo minimal changes in relative orientation and in packing, thereby acting as hinges, or flexible joints. The exchange rates of the indole proton of Trpbeta37(C3) were measured by nuclear magnetic resonance spectroscopy, in both deoxygenated and ligated hemoglobin. The results indicate that, at 15 degrees C, the exchange rate is increased from 9.0. 10(-6) to 3.3. 10(-4) s(-1) upon ligand binding to hemoglobin. This change suggests that the structural units at the hinge regions of the alpha1beta2/alpha2beta1 interfaces containing Trpbeta37(C3) are specifically stabilized in unligated hemoglobin, and experience a change in structural free energy of approximately 4 kcal/(mol tetramer) upon ligand binding. Therefore, the hinge regions of the alpha1beta2/alpha2beta1 interfaces could play a role in the transmission of free energy through the hemoglobin molecule during its allosteric transition.

Amino Acid Substitution↗

PubMed2001

The heme-globin and dimerization equilibria of recombinant human hemoglobins carrying site-specific beta chains mutations.

The heme-globin and dimer-tetramer equilibria of ferric recombinant human hemoglobins with site-specific beta chain mutations at the heme pocket or at either the a1beta1 or the alpha1beta2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the beta chain heme and does not affect the dimer-tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of beta chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of beta chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, beta chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.

Albumins↗

PubMed1999

Cysteines beta93 and beta112 as probes of conformational and functional events at the human hemoglobin subunit interfaces.

Three variants of tetrameric human hemoglobin, with changes at the alpha1beta2/alpha2beta1-interface, at the alpha1beta1/alpha2beta2-interface, and at both interfaces, have been constructed. At alpha1beta2/alpha2beta1-interface the beta93 cysteine was replaced by alanine (betaC93A), and at the alpha1beta1/alpha2beta2-interface the beta112 cysteine was replaced by glycine (betaC112G). The alpha1beta2 interface variant, betaC93A, and the alpha1beta1/alpha1beta2 double mutant, beta(C93A+C112G), were crystallized in the T-state, and the structures determined at 2. 0 and 1.8 A resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the beta112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of betaC112G are essentially those of human hemoglobin A. At the beta93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of beta146His, weakening the important interaction of this residue with beta94Asp. As a result, when Cl- is present in the solution, at a concentration 100 mM, the Bohr effect of the two mutants carrying the beta93Cys-->Ala substitution, betaC93A and beta(C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between beta94Asp and Cl- in the salt link with beta146His in T-state hemoglobin. These results point to an interplay between the betaHis146-betaAsp94 salt bridge and the Cl- in solution regulated by the Cys present at position beta93, indicating yet another role of beta93 Cys in the regulation of hemoglobin function.

Amino Acid Substitution↗

PubMed1998

Properties of human hemoglobins with increased polarity in the alpha- or beta-heme pocket. Carbonmonoxy derivatives.

The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A0 conformer in the IR spectrum, (iii) increased CO dissociation rates. The spectroscopic data indicate that for the carbonmonoxy derivatives, the Val --> Thr mutation has a larger conformational effect on the beta-subunits than on the alpha-subunits. This is at variance with the deoxy derivatives where the conformational modification was larger in the heme pocket of the alpha-subunit (Cupane, A., Leone, M., Militello, V., Friedman, R. K., Koley, A. P., Vasquez, G. P., Brinigar, W. S., Karavitis, M., and Fronticelli, C. (1997) J. Biol. Chem. 272, 26271-26278). These effects are attributed to a different electrostatic interaction between Ogamma of Thr(E11) and the bound CO molecule. Molecular analysis indicates a more favorable interaction of the bound CO with Thr Ogamma in the beta-subunit heme pocket.

Carboxyhemoglobin↗

PubMed1998

Human carboxyhemoglobin at 2.2 A resolution: structure and solvent comparisons of R-state, R2-state and T-state hemoglobins.

The three-dimensional structure and associated solvent of human carboxyhemoglobin at 2.2 A resolution are compared with other R-state and T-state human hemoglobin structures. The crystal form is isomorphous with that of the 2.7 A structure of carboxyhemoglobin reported earlier [Baldwin (1980). J. Mol. Biol. 136, 103-128], whose coordinates were used as a starting model, and with the 2.2 A structure described in an earlier report [Derewenda et al. (1990). J. Mol. Biol. 211, 515-519]. During the course of the refinement, a natural mutation of the alpha-subunit, A53S, was discovered that forms a new crystal contact through a bridging water molecule. The protein structure shows a significant difference between the alpha and beta heme geometries, with Fe-C-O angles of 125 and 162 degrees, respectively. The carboxyhemoglobin is compared with other fully ligated R-state human hemoglobins [Baldwin (1980). J. Mol. Biol. 136, 103-128; Shaanan (1983). J. Mol. Biol. 195, 419-422] with the R2-state hemoglobin [Silva et al. (1992). J. Biol. Chem. 267, 17248-17256] and with T-state deoxyhemoglobin [Fronticelli et al. (1994). J. Biol. Chem. 269, 23965-23969]. The structure is similar to the earlier reported R-state structures, but there are differences in many side-chain conformations, the associated water structure and the presence and the position of a phosphate ion. The quaternary changes between the R-state carboxyhemoglobin and the R2-state and T-state structures are in general consistent with those reported in the earlier structures. The location of 238 water molecules and a phosphate ion in the carboxyhemoglobin structure allows the first comparison of the solvent structures of the R-state and T-state structures. Distinctive hydration patterns for each of the quaternary structures are observed, but a number of conserved water molecule binding sites are found that are independent of the conformational state of the protein.

Carboxyhemoglobin↗

PubMed1998

Conformation of the sebacyl beta1Lys82-beta2Lys82 crosslink in T-state human hemoglobin.

The crystal structure of human T state hemoglobin crosslinked with bis(3,5-dibromo-salicyl) sebacate has been determined at 1.9 A resolution. The final crystallographic R factor is 0.168 with root-mean-square deviations (RMSD) from ideal bond distance of 0.018 A. The 10-carbon sebacyl residue found in the beta cleft covalently links the two betaLys82 residues. The sebacyl residue assumes a zigzag conformation with cis amide bonds formed by the NZ atoms of betaLys82's and the sebacyl carbonyl oxygens. The atoms of the crosslink have an occupancy factor of 1.0 with an average temperature factor for all atoms of 34 A2. An RMSD of 0.27 for all CA's of the tetramer is observed when the crosslinked deoxyhemoglobin is compared with deoxyhemoglobin refined by using a similar protocol, 2HHD [Fronticelli et al. J. Biol. Chem. 269: 23965-23969, 1994]. Thus, no significant perturbations in the tertiary or quaternary structure are introduced by the presence of the sebacyl residue. However, the sebacyl residue does displace seven water molecules in the beta cleft and the conformations of the beta1Lys82 and beta2Lys82 are altered because of the crosslinking. The carbonyl oxygen that is part of the amide bond formed with the NZ of beta2Lys82 forms a hydrogen bond with side chain of beta2Asn139 that is in turn hydrogen-bonded to the side chain of beta2Arg104. A comparison of the observed conformation with that modeled [Bucci et al. Biochemistry 35:3418-3425, 1996] shows significant differences. The differences in the structures can be rationalized in terms of compensating changes in the estimated free-energy balance, based on differences in exposed surface areas and the observed shift in the side-chain hydrogen-bonding pattern involving beta2Arg104, beta2Asn139, and the associated sebacyl carbonyl group.

Cross-Linking Reagents↗

PubMed1998

Alpha-subunit oxidation in T-state crystals of a sebacyl cross-linked human hemoglobin with unusual autoxidation properties.

In the crystal structure of human T-state hemoglobin with a sebacyl residue cross-linking the two beta-subunit Lys82,s (DecHb), the Fe atoms of the alpha-subunit hemes are found to be oxidized with a water molecule bound. The three-dimensional structure and heme geometries were compared to those of deoxyhemoglobin and other partially and fully oxidized hemoglobins [R. Liddington, Z. Derewenda, E. Dodson, R. Hubbard, G. Dodson, J. Mol. Biol. 228 (1992) 551]. The heme geometries of the alpha-subunits are consistent with those observed in oxidized structures. The proximal histidines of the alpha-subunits move toward the heme plane shifting the F-helix and FG-corner in a manner observed for partially oxidized human hemoglobin. This supports the hypothesis that these perturbations may precede the T- to R-state transition. Circular dichroism studies comparing DecHb and natural human hemoglobin in the deoxy and CO ligated forms confirm that the conformations of the deoxy forms are identical, but the ligated forms have slight differences in the solution structures. DecHb is found to be more resistant to autoxidation than natural hemoglobin. The time course of autoxidation of DecHb shows that it is virtually absent for the first 1500 min followed by a rapid increase. Thus, the discovery of the oxidation of the alpha-subunits in the deoxy-crystals is quite unexpected. The data confirm that ligation of the alpha-subunits precedes that of the beta-subunits. This may suggest a low ligand affinity of the alpha-diligated form of hemoglobin.

Circular Dichroism↗

PubMed1997

Modification of alpha-chain or beta-chain heme pocket polarity by Val(E11) --> thr substitution has different effects on the steric, dynamic, and functional properties of human recombinant hemoglobin. Deoxy derivatives.

The dynamic and functional properties of mutant deoxyhemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified alpha chain and beta chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the alpha chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the beta chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified alpha chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the alpha chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified beta chains is not observed by x-ray crystallography.

Carbon Monoxide↗

PubMed1997

Assembly of human hemoglobin. Studies with Escherichia coli-expressed alpha-globin.

The alpha-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native beta-chains. The functional and structural properties of the expressed alpha-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl- and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the alpha1beta1 and alpha1beta2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed alpha-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the alpha-globin refolds and incorporates heme in the absence of the partner beta-chain. Although the CO binding kinetics of recombinant alpha-chains were the same as that of native alpha-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant alpha-chain assumes alternating conformations in the absence of beta-chain and indicate that the isolated alpha-chain exhibits a higher degree of conformational flexibility than the alpha-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed alpha-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.

Adult↗

PubMed1997

Introduction of negative charges to a crosslinked hemoglobin: lack of effect on plasma half time.

Intramolecularly crosslinked hemoglobins do not dissociate into alpha 1 beta 1 dimers. As a result, they escape glomerular filtration and have plasma half times of 4 hours. This value is shorter than for albumin (5.2 hours) with similar molecular weight but higher negative charge. The present study was done to determine if increased negative charge on a hemoglobin covalently crosslinked with bis (3,5-dibromosalicyl) sebacate would lengthen its plasma half time. Negative charge was introduced by acylation with succinic anhydride. The product had a higher negative charge; however, plasma half time was not increased. A larger fraction of the succinylated material was excreted in the urine suggesting molecular instability.

Animals↗

PubMed1996

Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin.

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.

Allosteric Regulation↗

PubMed1996

Crystallographic, molecular modeling, and biophysical characterization of the valine beta 67 (E11)-->threonine variant of hemoglobin.

The crystal structure of the mutant deoxyhemoglobin in which the beta-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry 32, 1235-1242] has been determined at 2.2 A resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal beta-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the N delta of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant beta-subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry 32, 13061-13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the beta-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry 32, 1235-1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the O gamma atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the beta-subunits. In the aquomet derivative there is a 6-fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the O gamma of Thr67(E11).

Biophysical Phenomena↗

PubMed1996

Stabilization of the tetrameric structure of human and bovine hemoglobins by pseudocrosslinking with muconic acid.

In previous studies mono-3,5-dibromosalicyl-fumarate was used to introduce an intramolecular crosslink (pseudo-crosslink) in the beta cleft between hemoglobin beta subunits. Sedimentation velocity analysis indicated that the product had a mean molecular weight indicating a tetramer with low dissociability. The product had a P50 higher than that of native hemoglobin and a plasma retention time in the rat of about 3 h, i.e., four times longer than untreated hemoglobin. However, the product contained a fraction which was rapidly eliminated in the urine and which had a short plasma half-time of about 20 min, indicating the presence of a dissociable fraction. We have attempted to further enhance the tetrameric stability of hemoglobin and prevent urine elimination by positioning a longer chain carboxylic acid than fumaric acid into the beta cleft. We reason that a longer molecule would allow for greater stabilizing interactions across the beta cleft. In the present study human and bovine hemoglobins were reacted with mono-3-5-dibromosalicyl muconate. Muconic acid is two carbons longer than fumaric acid. The products were acylated at the beta 82 (human) and beta 81 (bovine) lysines of the beta-cleft and had a low degree of dissociability. For reasons not presently understood, urine excretion was high and plasma half-time was not increased above that of untreated hemoglobin. In conclusion, it appears that only covalently crosslinked hemoglobins which are completely nondissociable tetramers escape filtration; tetramers with any degree of dissociability into dimers are filterable.

Amino Acid Sequence↗

PubMed1995

Allosteric modulation by tertiary structure in mammalian hemoglobins. Introduction of the functional characteristics of bovine hemoglobin into human hemoglobin by five amino acid substitutions.

Bovine erythrocytes do not contain 2,3-diphosphoglycerate, the principal allosteric effector of human hemoglobin. Bovine hemoglobin has a lower oxygen affinity than human hemoglobin and is regulated by physiological concentrations of chloride (Fronticelli, C., Bucci, E., and Razynska, A. (1988) J. Mol. Biol. 202, 343-348). It has been proposed that the chloride regulation in bovine hemoglobin is introduced by particular amino acid residues located in the amino-terminal region of the A helix and in the E helix of the beta subunits (Fronticelli, C. (1990) Biophys. Chem. 37, 141-146). In accordance with this proposal we have constructed two mutant human hemoglobins, beta(V1M+H2deleted+T4I+P5A) and beta(V1M+H2deleted+T4I+P5A+A76K). These are the residues present at the proposed locations in bovine hemoglobin except for isoleucine at position 4. Oxygen binding studies demonstrate that these mutations have introduced into human hemoglobin the low oxygen affinity and chloride sensitivity of bovine hemoglobin and reveal the presence of a previously unrecognized allosteric mechanism of oxygen affinity regulation where all the interactions responsible for the lowered affinity and chloride binding appear to be confined to individual beta subunits.

Allosteric Regulation↗

PubMed1995

Preparative and analytical applications of immobilized haemoglobin.

New applications of haemoglobin immobilized on Sepharose 4B are proposed for the removal of cell-free, potentially toxic haemoglobin from either natural biological specimens or artificial haemoglobin-containing systems (e.g., blood substitutes, haemosomes) to be employed for biomedical purposes. In a model study, an affinity column of immobilized haemoglobin was used to remove free haemoglobin from blood serum in which controlled haemolysis had been induced. The affinity column retains all the free haemoglobin, does not retain the haemoglobin-haptoglobin complex(es) and leaves the composition of the serum samples unaltered. When immobilized met-haemoglobin is used, haem is transferred readily to albumin, with which it forms a complex. This observation on the one hand shows that immobilized oxyhaemoglobin should be preferred for preparative purposes, and on the other opens the way to the characterization of the haem transfer reaction to albumin by means of immobilized met-haemoglobin. This reaction is difficult to study in solution owing to the overlap of the met-haemoglobin and methaemalbumin spectra.

Cell-Free System↗

PubMed1995

Dynamic properties of some beta-chain mutant hemoglobins.

The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some beta-chain mutant hemoglobins is studied in the temperature range 300-10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the over-all bandwidth are singled out with this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wild-type and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme-CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the "polar isosteric" beta Val-67(E11)-->Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue beta Lys-144(Hc1) at the C-terminal and residue beta Cys-112(G14) at the alpha 1 beta 1 interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the beta-N-terminal and at the alpha 1 beta 2 interface have no effect on the dynamic properties of the same heme pocket.

Base Sequence↗