scholarly journals Structural and functional effects of selective chemical modifications of Scapharca inaequivalvis haemoglobins in relation to their unique assembly

1987 ◽  
Vol 241 (2) ◽  
pp. 499-504 ◽  
Author(s):  
A Boffi ◽  
M Gattoni ◽  
R Santucci ◽  
P Vecchini ◽  
F Ascoli ◽  
...  
Keyword(s):  
Functional Properties ◽  
Oxygen Affinity ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Oxygen Binding ◽  
Thiol Groups ◽  
Functional Roles ◽  
Dimeric Unit ◽  
Structural And Functional Properties ◽  
Thiol Reagent

The structural and functional roles of lysyl and thiol groups in the dimeric (HbI) and tetrameric (HbII) haemoglobins from the mollusc Scapharca inaequivalvis have been assessed. In these haemoglobins a unique mode of assembly (the haem-carrying E and F helices form the intersubunit contact of the dimeric unit) is associated with co-operative oxygen binding. Extensive acylation is accompanied by significant haem oxidation. Modification of one or two lysyl residues per chain (corresponding to approximately 20% of the total residues) does not affect the structural and functional properties of both haemoglobins, in line with the proposal that the intersubunit contacts are rich in hydrophobic residues. The modification of the thiol groups does not influence the state of association in both HbI and HbII, despite the location of the cysteine residue common to all polypeptide chains in the vicinity of the major intersubunit contact. The effect on the functional properties depends on the size of the thiol reagent: p-chloromercuribenzoate and phenylmercuric acetate increase the oxygen affinity about 20-fold, but iodoacetamide and mercuric chloride have no effect. Moreover, electrophoresis experiments indicate that p-chloromercuribenzoate is bound in a co-operative fashion, the degree of co-operativity being much higher in the dimeric HbI. Thus, only in HbII are intermediates containing substoichiometric amounts of p-chloromercuribenzoate formed in significant amounts. Their oxygen binding properties show that reaction of only one thiol group/tetramer suffices to alter the oxygen affinity of the molecule.

scholarly journals Adult and fetal haemoglobin J-Sardegna [α50(CE8)His→Asp]: functional and molecular modelling studies

2000 ◽  
Vol 346 (1) ◽  
pp. 193-199 ◽  
Author(s):  
Marcella CORDA ◽  
Maria C. DE ROSA ◽  
Maria G. PELLEGRINI ◽  
Maria T. SANNA ◽  
Alessandra OLIANAS ◽  
...  
Keyword(s):  
Functional Properties ◽  
Computational Modelling ◽  
Oxygen Affinity ◽  
Oxygen Binding ◽  
Structural Level ◽  
Fetal Haemoglobin ◽  
Significant Difference ◽  
Molecular Modelling Studies ◽  
Modelling Studies ◽  
2,3 Dpg

Haemoglobin (Hb) J-Sardegna [α50(CE8)His → Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 °C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 °C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the α1-β1 interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [α50(CE8)His → Arg] at the same position.

scholarly journals Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin

2007 ◽  
Vol 402 (1) ◽  
pp. 143-151 ◽  
Author(s):  
Tao Hu ◽  
Belur N. Manjula ◽  
Dongxia Li ◽  
Michael Brenowitz ◽  
Seetharama A. Acharya
Keyword(s):  
Functional Properties ◽  
Oxygen Affinity ◽  
Direct Consequence ◽  
Molecular Properties ◽  
Cross Linking ◽  
Hydrodynamic Drag ◽  
Cross Link ◽  
Central Cavity ◽  
Structural And Functional Properties ◽  
Cross Links

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an αα-fumaryl cross-link at Lys-99(α). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)6-αα-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)6-Hb. Perturbation of the haem microenvironment and the α1β2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)6-Hb and (propyl-PEG5K)6-αα-Hb sediment as stable dimeric and tetrameric molecules, respectively. The ββ-succinimidophenyl PEG-2000 cross-link at Cys-93(β) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the αα-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. αα-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.

scholarly journals Rat α-foetoprotein. Purification, physicochemical characterization, oestrogen-binding properties and chemical modification of the thiol group

1978 ◽  
Vol 175 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Vera Versée ◽  
André O. Barel
Keyword(s):  
Chemical Modification ◽  
Binding Site ◽  
Physicochemical Characterization ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Sulphonic Acid ◽  
Electrophoretic Variants ◽  
Thiol Reagent ◽  
Displacement Experiments

1. Rat α-foetoprotein, an oestrogen-binding foetal globulin, was isolated in large quantities from amniotic fluid and serum by preparative electrophoresis on polyacrylamide slab gels or by chromatography on an immunoadsorbent column. Subsequently the two electrophoretic forms of this protein were separated by electrophoresis on the same medium. 2. Both forms were found to show identical binding with oestradiol. From the extrinsic fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonic acid it was shown that the polarity of the binding site is practically identical for both forms. One residue of tryptophan was determined for both forms. The two electrophoretic variants display the same amount of secondary structure as demonstrated by circular dichroism. 3. The affinity of total α-foetoprotein for oestradiol as a function of pH was studied by using a Sephadex G-25 gel-equilibration method. Maximal binding occurred at pH8.5. Only a fractional number of binding sites per molecule could be measured at pH7.4, whereas at higher pH the number of sites was very close to unity. There was no significant effect of pH on the value of the association constant (Ka=4.3×107±1.2×107m−1). 4. Displacement experiments of bound labelled oestradiol with various steroids have permitted investigation of the specificity of α-foetoprotein. This foetal globulin binds rather strongly compounds that display the rigid structure of the oestratriene skeleton (oestradiol, oestrone). Diminished binding for diethylstilboestrol and a diethylstilboestrol affinity label was observed. No binding was measured with a more flexible structure such as hexoestrol [4,4′-(1,2-diethylethane-1,2-diyl)bisphenol]. 5. Chemical modification of cysteine residues of α-foetoprotein with two alkylating reagents [iodoacetic acid and 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid] has very little effect on the oestrogen binding. It is suggested that the oestrogen-binding site does not contain a cysteine residue. From the kinetics of alkylation and from the fluorescence properties of the chemically bound thiol reagent 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid], it was demonstrated that the very-slow-reacting thiol group is probably located in a non-polar region of the molecule.

scholarly journals Circe's haemoglobins, pig–human hybrids: functional characterization and structural considerations

1998 ◽  
Vol 335 (2) ◽  
pp. 211-216 ◽  
Author(s):  
Maria T. SANNA ◽  
Bruno GIARDINA ◽  
Mariagiuseppina PELLEGRINI ◽  
Alessandra OLIANAS ◽  
Irene MESSANA ◽  
...  
Keyword(s):  
Thermodynamic Properties ◽  
Structural Properties ◽  
Functional Properties ◽  
Functional Characterization ◽  
Oxygen Affinity ◽  
Oxygen Binding ◽  
Β Chain

We report the isolation and the functional characterization of α and β chains from pig (Sus scropha domesticus) haemoglobin, as well as of the pig–human hybrid haemoglobins, α2hβ2p and α2pβ2h (i.e. Circe's haemoglobins), obtained by mixing the purified α and β pig chains respectively with the corresponding partner human chains. Their functional properties have been compared with those of both parental haemoglobins in order to obtain information on the role of the different subunits and of their inter-relationships, both at the structural and functional levels. The results indicate that the functional properties of both hybrids are closer to those of the parental haemoglobin that provides the β chains, confirming the major role of the β chains in determining the oxygen affinity and the modulation mechanisms of the tetrameric molecule. This is supported by the thermodynamic properties, since the very low ΔH of oxygen binding that characterizes pig haemoglobin and the α2hβ2p hybrid haemoglobin may be taken as the reflection of specific structural properties of pig β chain.

Quaternary structure of Limulus polyphemus hemocyanin

1978 ◽  
Vol 36 (2) ◽  
pp. 176-177
Author(s):  
T. Wichertjes ◽  
E.J. Kwak ◽  
E.F.J. Van Bruggen
Keyword(s):  
Electron Microscopy ◽  
Functional Properties ◽  
Horseshoe Crab ◽  
Temperature Jump ◽  
Quaternary Structure ◽  
Crystallographic Analysis ◽  
Limulus Polyphemus ◽  
Oxygen Binding ◽  
X Ray ◽  
Intact Molecule

Hemocyanin of the horseshoe crab (Limulus polyphemus) has been studied in nany ways. Recently the structure, dissociation and reassembly was studied using electron microscopy of negatively stained specimens as the method of investigation. Crystallization of the protein proved to be possible and X-ray crystallographic analysis was started. Also fluorescence properties of the hemocyanin after dialysis against Tris-glycine buffer + 0.01 M EDTA pH 8.9 (so called “stripped” hemocyanin) and its fractions II and V were studied, as well as functional properties of the fractions by NMR. Finally the temperature-jump method was used for assaying the oxygen binding of the dissociating molecule and of preparations of isolated subunits. Nevertheless very little is known about the structure of the intact molecule. Schutter et al. suggested that the molecule possibly consists of two halves, combined in a staggered way, the halves themselves consisting of four subunits arranged in a square.

Structural and functional properties of Escherichia coli-derived nucleoplasmin A comparative study of recombinant and natural proteins

2001 ◽  
Vol 268 (6) ◽  
pp. 1739-1748
Author(s):  
Aitor Hierro ◽  
Jesus M. Arizmendi ◽  
Javier De Las Rivas ◽  
M. Angeles Urbaneja ◽  
Adelina Prado ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Functional Properties ◽  
Structural And Functional Properties
Sign in / Sign up

Export Citation Format

Share Document