The Experts below are selected from a list of 3201 Experts worldwide ranked by ideXlab platform
Theodore W Randolph - One of the best experts on this subject based on the ideXlab platform.
-
effects of tween 80 and sucrose on acute short term stability and long term storage at 20 c of a Recombinant Hemoglobin
Journal of Pharmaceutical Sciences, 1998Co-Authors: Bruce A. Kerwin, Martin C Heller, Steven H Levin, Theodore W RandolphAbstract:The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to Recombinant Hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze–thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze–thaw induced aggregation. In contrast to the acute stability studies, long-term storage at − 20 °C induced aggregation and metHemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested metHemoglobin formation during the long-term − 20 °C storage. Specific binding of Tween 80 to the Hemoglobin was not observed using 16–doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the Hemoglobin from reaching the air–liquid interface or the liquid– surface interfaces. The reduction in metHemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536–6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.
-
effects of tween 80 and sucrose on acute short term stability and long term storage at 20 c of a Recombinant Hemoglobin
Journal of Pharmaceutical Sciences, 1998Co-Authors: Bruce A. Kerwin, Steven H Levin, Martin Heller, Theodore W RandolphAbstract:The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to Recombinant Hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze-thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze-thaw induced aggregation. In contrast to the acute stability studies, long-term storage at -20 degrees C induced aggregation and metHemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested metHemoglobin formation during the long-term -20 degrees C storage. Specific binding of Tween 80 to the Hemoglobin was not observed using 16-doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the Hemoglobin from reaching the air-liquid interface or the liquid-surface interfaces. The reduction in metHemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536-6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.
-
Effects of Tween 80 and Sucrose on Acute Short-Term Stability and Long-Term Storage at − 20 C of a Recombinant Hemoglobin
Journal of Pharmaceutical Sciences, 1998Co-Authors: Bruce A. Kerwin, Steven H Levin, Martin Heller, Theodore W RandolphAbstract:The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to Recombinant Hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze–thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze–thaw induced aggregation. In contrast to the acute stability studies, long-term storage at − 20 °C induced aggregation and metHemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested metHemoglobin formation during the long-term − 20 °C storage. Specific binding of Tween 80 to the Hemoglobin was not observed using 16–doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the Hemoglobin from reaching the air–liquid interface or the liquid– surface interfaces. The reduction in metHemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536–6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.
-
Effects of Phase Separating Systems on Lyophilized Hemoglobin
Journal of Pharmaceutical Sciences, 1996Co-Authors: Martin Heller, John F. Carpenter, Theodore W RandolphAbstract:Polymer liquid-liquid two-phase systems offer a unique opportunity to study the mechanisms of protein stabilization during freezing and freeze-drying. Fourier transform infrared spectroscopy was used to monitor the structural integrity of Recombinant Hemoglobin frozen and lyophilized in the separated phases of a polyethylene glycol (PEG)-dextran system. Protein in each phase of an equilibrated biphasic PEG-dextran system experiences similar levels of structural protection against freezing stresses despite large differences in polymer concentration. This result further demonstrates previous suggestions that proteins are protected during freezing by the preferential exclusion mechanism. There are, however, distinct differences in the level of structural protection that polymers in equilibrium phases provide to proteins during lyophilization, emphasizing that the mechanisms of protein protection during freezing and drying are fundamentally different. In addition, we provide evidence that phase separation per se occurring during the course of the lyophilization cycle can be detrimental to the structural stability of a protein.
Josée Pagnier - One of the best experts on this subject based on the ideXlab platform.
-
Stable octameric structure of Recombinant Hemoglobin α2β283 Gly→Cys
Protein Science, 2003Co-Authors: Christophe Fablet, Josée Pagnier, Brian N. Green, Chien Ho, Michael C Marden, Véronique Baudin-creuzaAbstract:We have engineered a Recombinant Hemoglobin (rHb βG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb βG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human Hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric βG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric βG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer βG83C was not modified after incubation with fresh plasma.
-
stable octameric structure of Recombinant Hemoglobin α2β283 gly cys
Protein Science, 2003Co-Authors: Christophe Fablet, Josée Pagnier, Brian N. Green, Michael C Marden, Veronique BaudincreuzaAbstract:We have engineered a Recombinant Hemoglobin (rHb βG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb βG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human Hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric βG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric βG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer βG83C was not modified after incubation with fresh plasma.
-
Functional Studies and Polymerization of Recombinant Hemoglobin Glu-α2β26(A3) → Val/Glu-7(A4) → Ala
Journal of Biological Chemistry, 1996Co-Authors: Sophie Lesecq, Michael C Marden, V. Baudin, Jean Kister, Claude Poyart, Josée PagnierAbstract:Abstract In Hemoglobin (Hb) S the hydrophobic mutated residue Val-β6(A3) (donor site) closely interacts with the hydrophobic side groups of Phe-β85(F1) and Leu-β88(F4) (EF pocket, acceptor site) of a neighboring tetramer, resulting in decreased solubility and polymerization of the deoxy-Hb. The β6(A3) residue is followed by two charged residues Glu-β7(A4) and Lys-β8(A5). This cluster has no attraction for the hydrophobic EF pocket. We have modified the β7(A4) residue next to the donor site Val-β6(A3), replacing the charged Glu by a hydrophobic Ala-(rHb βE6V/E7A). The single mutant Glu-β7 → Ala-(rHb βE7A) was also engineered. Both rHbs exhibit a heat instability and an increased oxygen affinity compared to Hb A and Hb S. There was a concentration dependence of the ligand binding properties (1-300 μM in heme) indicating an increased amount of dimers relative to Hb A. The deoxy form of rHb βE6V/E7A polymerizes in vitro, with a decreased rate of polymer formation relative to Hb S, while the single mutant βE7A does not polymerize in the same experimental conditions. The Glu-β7(A4) → Ala substitution does not increase the hydrophobic interaction between donor and acceptor site. We speculate that the loss of the normal saline bridge between Glu-β7(A4) and Lys-β132(H10) leads to an increased flexibility of the A helix and may account for the difference of the polymerization for this Hb S mutant.
-
functional studies and polymerization of Recombinant Hemoglobin glu α2β26 a3 val glu 7 a4 ala
Journal of Biological Chemistry, 1996Co-Authors: Sophie Lesecq, Michael C Marden, V. Baudin, Jean Kister, Claude Poyart, Josée PagnierAbstract:Abstract In Hemoglobin (Hb) S the hydrophobic mutated residue Val-β6(A3) (donor site) closely interacts with the hydrophobic side groups of Phe-β85(F1) and Leu-β88(F4) (EF pocket, acceptor site) of a neighboring tetramer, resulting in decreased solubility and polymerization of the deoxy-Hb. The β6(A3) residue is followed by two charged residues Glu-β7(A4) and Lys-β8(A5). This cluster has no attraction for the hydrophobic EF pocket. We have modified the β7(A4) residue next to the donor site Val-β6(A3), replacing the charged Glu by a hydrophobic Ala-(rHb βE6V/E7A). The single mutant Glu-β7 → Ala-(rHb βE7A) was also engineered. Both rHbs exhibit a heat instability and an increased oxygen affinity compared to Hb A and Hb S. There was a concentration dependence of the ligand binding properties (1-300 μM in heme) indicating an increased amount of dimers relative to Hb A. The deoxy form of rHb βE6V/E7A polymerizes in vitro, with a decreased rate of polymer formation relative to Hb S, while the single mutant βE7A does not polymerize in the same experimental conditions. The Glu-β7(A4) → Ala substitution does not increase the hydrophobic interaction between donor and acceptor site. We speculate that the loss of the normal saline bridge between Glu-β7(A4) and Lys-β132(H10) leads to an increased flexibility of the A helix and may account for the difference of the polymerization for this Hb S mutant.
-
The Role of Proline β5(A2) in the Functional Properties of Human Adult Hemoglobin
Hemoglobin, 1996Co-Authors: V. Baudin, Jean Kister, Claude Poyart, G. Caron, V. Jonval, Josée PagnierAbstract:The replacement of β35(A2)Pro by Arg in Hb Warwickshire appears to be without an effect on the functional properties of human Hb A, despite adding two external positive charges close to the central cavity of the Hemoglobin tetramer, along the dyad axis. To clarify the role of this portion of the molecule involved in oxygen-linked anion binding, we have engineered the Recombinant Hemoglobin α2β5(A2)Pro→Ala [rHD β5(A2)Pro→Ala]. The rHb β5(A2)Pro→Ala exhibits an increased oxygen affinity compared to Hb A, with normal heterotropic effects in standard conditions. The increased oxygen affinity may be attributed to the absence of proline, which would render the A helix more flexible, thus destabilizing the T structure. The normal functional properties of Hb Warwickshire may be due to the regulation of oxygen affinity by electrostatic effects involving diffusible anions not bound to any specific site.
Bruce A. Kerwin - One of the best experts on this subject based on the ideXlab platform.
-
Acute and Long-Term Stability Studies of Deoxy Hemoglobin and Characterization of Ascorbate-Induced Modifications
Journal of Pharmaceutical Sciences, 1999Co-Authors: Bruce A. Kerwin, Julie Lippincott, Edward Hess, Izydor Apostol, Joseph Levine, Antony J. Mathews, Michael J. Akers, Camille Moore‐einsel, Jeffrey E. Etter, Patricia Revilla‐sharpAbstract:The reaction of ascorbate with Recombinant Hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the Hemoglobin solution to
-
effects of tween 80 and sucrose on acute short term stability and long term storage at 20 c of a Recombinant Hemoglobin
Journal of Pharmaceutical Sciences, 1998Co-Authors: Bruce A. Kerwin, Martin C Heller, Steven H Levin, Theodore W RandolphAbstract:The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to Recombinant Hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze–thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze–thaw induced aggregation. In contrast to the acute stability studies, long-term storage at − 20 °C induced aggregation and metHemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested metHemoglobin formation during the long-term − 20 °C storage. Specific binding of Tween 80 to the Hemoglobin was not observed using 16–doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the Hemoglobin from reaching the air–liquid interface or the liquid– surface interfaces. The reduction in metHemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536–6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.
-
effects of tween 80 and sucrose on acute short term stability and long term storage at 20 c of a Recombinant Hemoglobin
Journal of Pharmaceutical Sciences, 1998Co-Authors: Bruce A. Kerwin, Steven H Levin, Martin Heller, Theodore W RandolphAbstract:The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to Recombinant Hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze-thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze-thaw induced aggregation. In contrast to the acute stability studies, long-term storage at -20 degrees C induced aggregation and metHemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested metHemoglobin formation during the long-term -20 degrees C storage. Specific binding of Tween 80 to the Hemoglobin was not observed using 16-doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the Hemoglobin from reaching the air-liquid interface or the liquid-surface interfaces. The reduction in metHemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536-6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.
-
Effects of Tween 80 and Sucrose on Acute Short-Term Stability and Long-Term Storage at − 20 C of a Recombinant Hemoglobin
Journal of Pharmaceutical Sciences, 1998Co-Authors: Bruce A. Kerwin, Steven H Levin, Martin Heller, Theodore W RandolphAbstract:The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to Recombinant Hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze–thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze–thaw induced aggregation. In contrast to the acute stability studies, long-term storage at − 20 °C induced aggregation and metHemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested metHemoglobin formation during the long-term − 20 °C storage. Specific binding of Tween 80 to the Hemoglobin was not observed using 16–doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the Hemoglobin from reaching the air–liquid interface or the liquid– surface interfaces. The reduction in metHemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536–6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.
-
Oxygen and ascorbate mediated modification of a Recombinant Hemoglobin
Techniques in Protein Chemistry, 1997Co-Authors: Bruce A. Kerwin, Julie Lippincott, Edward Hess, Ray Kaiser, Izydor ApostolAbstract:Publisher Summary In the reduced state the iron center of each of the four heme group in Recombinant Hemoglobin, rHb1.1, reversibly binds molecular oxygen. Upon binding oxygen the Hemoglobin can autoxidize forming metHb, which is incapable of binding oxygen. The metHb can be reduced back to the ferrous state using ascorbate and reduced oxygen conditions. In the presence of oxygen, however, ascorbate reacts with molecular oxygen to form dehydroascorbate and superoxide anion. The dehydroascorbate can undergo hydrolytic ring rupture to form 2,3-diketogulonic acid that in turn can react further with oxygen forming additional byproducts, which may modify proteins. One of the more prevalent protein modifications detected has been carboxymethylation of lysine groups to form Nɛ-(carboxymethyl)lysine. Although it is known that the superoxide reacts with and oxidizes the Hemoglobin, little is known concerning the modification of the Hemoglobin by dehydroascorbate and its byproducts. Subsequent interest lies in determining how rHb1.1 is modified in the presence of dehydroascorbate as well as in the presence of ascorbate and oxygen. The location of the modification has been determined using trypsin mapping. Following dehydroascorbate modification of deoxy-rHb, the main β-globin peak and the lagging shoulder of the p-globin are purified from reverse phase HPLC and mapped with trypsin. The map of the unmodified P-globin has been shown in this chapter and is not different from that of P-globin not exposed to ascorbate.
Juliette T. J. Lecomte - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of the heme–histidine cross-link in cyanobacterial Hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002
JBIC Journal of Biological Inorganic Chemistry, 2004Co-Authors: David A. Vuletich, Christopher J. Falzone, Syna A. Kuriakose, Juliette T. J. LecomteAbstract:The Recombinant product of the Hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the ^1H, ^15N, and ^13C NMR spectroscopy experiments demonstrating that the Recombinant Hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis Hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis Hemoglobin suggests that it constitutes a noteworthy feature of Hemoglobin in non-N_2-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis Hemoglobin, the unreacted His117 imidazole had a normal p K _a whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.
-
Novel histidine-heme covalent linkage in a Hemoglobin.
Journal of the American Chemical Society, 2002Co-Authors: A. Daniel Jones, Juliette T. J. LecomteAbstract:When treated with dithionite at neutral pH, the Recombinant Hemoglobin from Synechocystis sp. PCC 6803 reconstituted with ferric heme undergoes a rapid chemical reaction resulting in the attachment of the heme group to the polypeptide chain. The nature of the cross-linked species was studied by NMR and mass spectral methods. 1H NMR data indicated that the 2-vinyl group was the reacting moiety of the heme. Mass spectrometry of pepsin digests located the site of attachment within a 12-mer at the C-terminal end of the protein. Homonuclear and 1H-15N NMR data identified the modified residue as His117, which underwent addition to the vinyl Calpha through the imidazole Nepsilon. Dithionite treatment of the globin reconstituted with Zn protoporphyrin IX sample did not lead to 2-vinyl group modification, suggesting that the chemical reduction of the heme iron facilitated the attachment.
-
The Solution Structure of the Recombinant Hemoglobin from the Cyanobacterium Synechocystis sp. PCC 6803 in its Hemichrome State
Journal of Molecular Biology, 2002Co-Authors: Christopher J. Falzone, Nancy L. Scott, Juliette T. J. LecomteAbstract:The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated Hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 Hemoglobin displays bis-histidine coordination of the iron ion. In addition, this protein is capable of covalently attaching a reactive histidine to the heme 2-vinyl group. The structure of the protein in the low-spin ferric state with intact vinyl substituents was solved by NMR methods. It was found that the structure differs from that of known truncated Hemoglobins primarily in the orientation of the E helix, which carries His46 (E10) as the distal ligand to the iron; the length and orientation of the F helix, which carries His70 (F8) as the proximal ligand to the iron; and the H-helix, which carries His117 (H16), the reactive histidine. Regions of enhanced flexibility include the short A helix, the loop connecting the E and F helices, and the last seven residues at the carboxy end. The structural data allowed for the rationalization of physical properties of the cyanobacterial protein, such as fast on-rate for small ligand binding, unstable apoprotein fold, and cross-linking ability. Comparison to the truncated Hemoglobin from the green alga Chlamydomonas eugametos also suggested how the endogenous hexacoordination affected the structure.
Michael C Marden - One of the best experts on this subject based on the ideXlab platform.
-
Recombinant Hemoglobin βG83C-F41Y
FEBS Journal, 2006Co-Authors: Corinne Vasseur-godbillon, Christophe Fablet, Michael C Marden, Sarata C. Sahu, Elisa Domingues, Janel L. Giovannelli, Tsuey Chyi Tam, Véronique Baudin-creuzaAbstract:We have engineered a stable octameric Hemoglobin (Hb) of molecular mass 129 kDa, a dimer of Recombinant Hemoglobin (rHb βG83C-F41Y) tetramers joined by disulfide bonds at the β83 position. One of the major problems with oxygen carriers based on acellular Hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb βG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb βG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 m−1·h−1 (observed time coefficient of 1 h at 1 mm glutathione) at 25 °C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 µm Hb and 25 °C. In addition, the octameric rHb βG83C-F41Y was resistant to potential reducing agents present in fresh plasma.
-
Recombinant Hemoglobin betag83c f41y
FEBS Journal, 2006Co-Authors: Corinne Vasseurgodbillon, Christophe Fablet, Sarata C. Sahu, Elisa Domingues, Janel L. Giovannelli, Tsuey Chyi Tam, Michael C MardenAbstract:We have engineered a stable octameric Hemoglobin (Hb) of molecular mass 129 kDa, a dimer of Recombinant Hemoglobin (rHb betaG83C-F41Y) tetramers joined by disulfide bonds at the beta83 position. One of the major problems with oxygen carriers based on acellular Hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb betaG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb betaG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 M(-1) x h(-1) (observed time coefficient of 1 h at 1 mM glutathione) at 25 degrees C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 microM Hb and 25 degrees C. In addition, the octameric rHb betaG83C-F41Y was resistant to potential reducing agents present in fresh plasma.
-
Recombinant Hemoglobin betag83c f41y an octameric protein
FEBS Journal, 2006Co-Authors: Corinne Vasseurgodbillon, Christophe Fablet, Michael C Marden, Sarata C. Sahu, Elisa Domingues, Janel L. Giovannelli, Tsuey Chyi Tam, Veronique BaudincreuzaAbstract:We have engineered a stable octameric Hemoglobin (Hb) of molecular mass 129 kDa, a dimer of Recombinant Hemoglobin (rHb PG83C-F41Y) tetramers joined by disulfide bonds at the β83 position. One of the major problems with oxygen carriers based on acellular Hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb bG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb PG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 M - 1 .h - 1 (observed time coefficient of 1 h at 1 mM glutathione) at 25 °C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 μM Hb and 25 °C. In addition, the octameric rHb PG83C-F41Y was resistant to potential reducing agents present in fresh plasma.
-
Stable octameric structure of Recombinant Hemoglobin α2β283 Gly→Cys
Protein Science, 2003Co-Authors: Christophe Fablet, Josée Pagnier, Brian N. Green, Chien Ho, Michael C Marden, Véronique Baudin-creuzaAbstract:We have engineered a Recombinant Hemoglobin (rHb βG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb βG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human Hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric βG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric βG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer βG83C was not modified after incubation with fresh plasma.
-
stable octameric structure of Recombinant Hemoglobin α2β283 gly cys
Protein Science, 2003Co-Authors: Christophe Fablet, Josée Pagnier, Brian N. Green, Michael C Marden, Veronique BaudincreuzaAbstract:We have engineered a Recombinant Hemoglobin (rHb βG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb βG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human Hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric βG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric βG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer βG83C was not modified after incubation with fresh plasma.