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Keywords: haemoglobin
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Articles
Journal:
Biochemical Journal
Biochem J (2014) 458 (3): e5–e7.
Published: 28 February 2014
... still requires specific validation. Finally, given the well-known function of 23BPG as a negative allosteric effector of haemoglobin [ 11 ], it would be reasonable to speculate the possible role of TIGAR in regulating oxygen binding to haemoglobin. Gerin et al. [ 7 ] discarded this possibility in...
Abstract
TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator] protein is known for its ability to inhibit glycolysis, shifting glucose consumption towards the pentose phosphate pathway to promote antioxidant protection of cancer cells. According to sequence homology and activity analyses, TIGAR was initially considered to be a fructose-2,6-bisphosphatase; it has thus received much attention in cancer cell metabolism, given its dependence on p53 and the key role of F26BP (fructose 2,6-bisphosphate) at modulating glycolysis and gluconeogenesis. However, in a rigorous study published in this issue of the Biochemical Journal , Gerin and colleagues report that recombinant TIGAR is a 23BPG (2,3-bisphosphoglycerate) phosphatase, although it also dephosphorylates other carboxylic acid-phosphate esters and, weakly, F26BP. As such, inhibition of endogenous TIGAR leads to a dramatic increase in cellular 23BPG, influencing F26BP to a lower extent that depends on the cellular context. These results challenge the currently held notion that TIGAR modulates glycolysis through decreasing F26BP, and opens a yet unrecognized function(s) for TIGAR-mediated 23BPG control of cellular metabolism in health and disease.
Articles
Ryan Stapley, Benjamin Y. Owusu, Angela Brandon, Marianne Cusick, Cilina Rodriguez, Marisa B. Marques, Jeffrey D. Kerby, Scott R. Barnum, Jordan A. Weinberg, Jack R. Lancaster, Jr, Rakesh P. Patel
Journal:
Biochemical Journal
Biochem J (2012) 446 (3): 499–508.
Published: 28 August 2012
...@uab.edu ). 24 4 2012 15 6 2012 21 6 2012 21 6 2012 © The Authors Journal compilation © 2012 Biochemical Society 2012 haemoglobin inflammation kinetics nitric oxide nitrite vasoconstriction Transfusion with RBCs (red blood cells) is the front-line therapy for...
Abstract
Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to RBCs (red blood cells). It has been suggested that these changes have potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion-related toxicities. However, the mechanisms linking RBC storage and toxicity remain unclear. In the present study we tested the hypothesis that storage of leucodepleted RBCs results in cells that inhibit NO (nitric oxide) signalling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from haemolysis or microparticles, our data indicate that the consumption rates of NO increased ~40-fold and NO-dependent vasodilation was inhibited 2–4-fold comparing 42-day-old with 0-day-old RBCs. These results are probably due to the formation of smaller RBCs with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBCs to affect NO formation via deoxygenated RBC-mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBCs did increase the rates of nitrite oxidation to nitrate in vitro . Significant loss of whole-blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBCs stored for >25 days, but not with younger RBCs. Collectively, these data suggest that increased rates of reactions between intact RBCs and NO and nitrite may contribute to mechanisms that lead to storage-lesion-related transfusion risk.
Includes: Supplementary data
Articles
Journal:
Biochemical Journal
Biochem J (2012) 441 (3): 843–852.
Published: 16 January 2012
...María T. Marcos-Almaraz; José A. Rodríguez-Gómez; José López-Barneo; Alberto Pascual Discovery of haemoglobin A expression outside of the erythroid cell lineage suggests that oxygen transport is the main, but not the unique, function of adult haemoglobin chains in mammals. The contribution of...
Abstract
Discovery of haemoglobin A expression outside of the erythroid cell lineage suggests that oxygen transport is the main, but not the unique, function of adult haemoglobin chains in mammals. The contribution of haemoglobin A to antioxidant defences has been proposed in the territories where it is expressed. Catecholaminergic cells rely on an active oxidative metabolism to accomplish their biological function, but are exposed to strong oxidative stress due to metabolism of catecholaminergic transmitters. We show in the present study that peripheral catecholaminegic cells express the α- and not the β-haemoglobin A chains, and that α-haemoglobin expression could modulate the antioxidant capabilities of these cells. We also show that α-haemoglobin overexpression in PC12 cells leads to a selective increase of tyrosine hydroxylase synthesis and activity. This is achieved by means of a reorganization of antioxidant defences, decreasing cytoplasmic glutathione peroxidase and superoxide dismutase, and increasing mitochondrial peroxidase. Moreover, α-haemoglobin induces a decrease in lipogenesis and increase in lipid degradation, situations that help save NAD(P)H and favour supply of acetyl-CoA to the tricarboxylic acid cycle and production of reducing equivalents in the cell. All of these results point to a role for α-haemoglobin as a regulator of catecholaminergic cell metabolism required for phenotype acquisition and maintenance.
Includes: Supplementary data
Articles
Journal:
Biochemical Journal
Biochem J (2011) 434 (3): 483–492.
Published: 24 February 2011
...Brandon J. Reeder; Dimitri A. Svistunenko; Michael T. Wilson Cytoglobin is a recently discovered hexa-co-ordinate haemoglobin that does not appear to function as a classical oxygen-binding protein. Its function is unknown and studies on the effects of changes in its expression have not decisively...
Abstract
Cytoglobin is a recently discovered hexa-co-ordinate haemoglobin that does not appear to function as a classical oxygen-binding protein. Its function is unknown and studies on the effects of changes in its expression have not decisively determined its role within the cell. In the present paper, we report that the protein is transformed from hexa-co-ordinate to penta-co-ordinate on binding a lipid molecule. This transformation occurs with the ferric oxidation state of the protein, but not the ferrous state, indicating that this process only occurs under an oxidative environment and may thus be related to redox-linked cell signalling mechanisms. Oleate binds to the protein in a 1:1 stoichiometry and with high affinity ( K d =0.7 μM); however, stopped-flow kinetic measurements yield a K d value of 110 μM. The discrepancy between these K d values may be rationalized by recognizing that cytoglobin is a disulfide-linked dimer and invoking co-operativity in oleate binding. The lipid-induced transformation of cytoglobin from hexa-co-ordinate to penta-co-ordinate does not occur with similar hexa-co-ordinate haemoglobins such as neuroglobin, and therefore appears to be a unique property of cytoglobin among the haemoglobin superfamily. The lipid-derived transformation may explain why cytoglobin has enhanced peroxidatic activity, converting lipids into various oxidized products, a property virtually absent from neuroglobin and much decreased in myoglobin. We propose that the binding of ferric cytoglobin to lipids and their subsequent transformation may be integral to the physiological function of cytoglobin, generating cell signalling lipid molecules under an oxidative environment.
Includes: Supplementary data
Articles
Louay Mardini, Jadwiga Gasiorek, Anna Derjuga, Lucie Carrière, Matthias Schranzhofer, Barry H. Paw, Prem Ponka, Volker Blank
Journal:
Biochemical Journal
Biochem J (2010) 432 (1): 145–151.
Published: 25 October 2010
...Louay Mardini; Jadwiga Gasiorek; Anna Derjuga; Lucie Carrière; Matthias Schranzhofer; Barry H. Paw; Prem Ponka; Volker Blank Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In...
Abstract
Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In the present study, we investigated the role of the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in MEL (mouse erythroleukaemia) cell differentiation. We found that treatment of HMBA (hexamethylene bisacetamide)-induced MEL cells with the ERK pathway inhibitor UO126 results in an increase in intracellular haem and haemoglobin levels. The transcript levels of the genes coding for β major -globin, the haem biosynthesis enzyme 5-aminolevulinate synthase 2 and the mitochondrial iron transporter mitoferrin 1 are up-regulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 treatment. With respect to iron uptake, we found that ERK inhibitor treatment led to an increase in both haem-bound and total iron. In contrast, treatment of MEL cells with the p38 MAPK pathway inhibitor SB202190 had the opposite effect, resulting in decreased globin expression, haem synthesis and iron uptake. Reporter assays showed that globin promoter and HS2 enhancer-mediated transcription was under the control of MAPKs, as inhibition of the ERK and p38 MAPK pathways led to increased and decreased gene activity respectively. Our present results suggest that the ERK1/2 and p38α/β MAPKs play antagonistic roles in HMBA-induced globin gene expression and erythroid differentiation. These results provide a novel link between MAPK signalling and the regulation of haem biosynthesis and iron uptake in erythroid cells.
Articles
Journal:
Biochemical Journal
Biochem J (2010) 425 (1): 257–264.
Published: 14 December 2009
...Dominic P. Byrne; Katarzyna Wawrzonek; Anna Jaworska; Andrew J. Birss; Jan Potempa; John W. Smalley The Gram-negative oral anaerobe Prevotella intermedia forms an iron(III) protoporphyrin IX pigment from haemoglobin. The bacterium expresses a 90 kDa cysteine protease, InpA (interpain A), a...
Abstract
The Gram-negative oral anaerobe Prevotella intermedia forms an iron(III) protoporphyrin IX pigment from haemoglobin. The bacterium expresses a 90 kDa cysteine protease, InpA (interpain A), a homologue of Streptococcus pyogenes streptopain (SpeB). The role of InpA in haemoglobin breakdown and haem release was investigated. At pH 7.5, InpA mediated oxidation of oxyhaemoglobin to hydroxymethaemoglobin [in which the haem iron is oxidized to the Fe(III) state and which carries OH − as the sixth co-ordinate ligand] by limited proteolysis of globin chains as indicated by SDS/PAGE and MALDI (matrix-assisted laser-desorption ionization)–TOF (time-of-flight) analysis. Prolonged incubation at pH 7.5 did not result in further haemoglobin protein breakdown, but in the formation of a haemoglobin haemichrome (where the haem Fe atom is co-ordinated by another amino acid ligand in addition to the proximal histidine residue) resistant to degradation by InpA. InpA-mediated haem release from hydroxymethaemoglobin–agarose was minimal compared with trypsin at pH 7.5. At pH 6.0, InpA increased oxidation at a rate greater than auto-oxidation, producing aquomethaemoglobin (with water as sixth co-ordinate ligand), and resulted in its complete breakdown and haem loss. Aquomethaemoglobin proteolysis and haem release was prevented by blocking haem dissociation by ligation with azide, whereas InpA proteolysis of haem-free globin was rapid, even at pH 7.5. Both oxidation of oxyhaemoglobin and breakdown of methaemoglobin by InpA were inhibited by the cysteine protease inhibitor E-64 [ trans -epoxysuccinyl- L -leucylamido-(4-guanidino)butane]. In summary, we conclude that InpA may play a central role in haem acquisition by mediating oxyhaemoglobin oxidation, and by degrading aquomethaemoglobin in which haem–globin affinity is weakened under acidic conditions.
Articles
Journal:
Biochemical Journal
Biochem J (2007) 403 (3): 391–395.
Published: 12 April 2007
...Radu Silaghi-Dumitrescu; Brandon J. Reeder; Peter Nicholls; Chris E. Cooper; Michael T. Wilson Ferryl (Fe(IV)=O) species are involved in key enzymatic processes with direct biomedical relevance; among others, the uncontrolled reactivities of ferryl Mb (myoglobin) and Hb (haemoglobin) have been...
Abstract
Ferryl (Fe(IV)=O) species are involved in key enzymatic processes with direct biomedical relevance; among others, the uncontrolled reactivities of ferryl Mb (myoglobin) and Hb (haemoglobin) have been reported to be central to the pathology of rhabdomyolysis and subarachnoid haemorrhage. Rapid-scan stopped-flow methods have been used to monitor the spectra of the ferryl species in Mb and Hb as a function of pH. The ferryl forms of both proteins display an optical transition with p K ∼4.7, and this is assigned to protonation of the ferryl species itself. We also demonstrate for the first time a direct correlation between Hb/Mb ferryl reactivity and ferryl protonation status, simultaneously informing on chemical mechanism and toxicity and with broader biochemical implications.
Articles
Journal:
Biochemical Journal
Biochem J (2007) 402 (1): 143–151.
Published: 25 January 2007
...Tao Hu; Belur N. Manjula; Dongxia Li; Michael Brenowitz; Seetharama A. Acharya 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...
Abstract
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.
Articles
Jacqueline Dunne, Alexis Caron, Patrick Menu, Abdu I. Alayash, Paul W. Buehler, Michael T. Wilson, Radu Silaghi-Dumitrescu, Beatrice Faivre, Chris E. Cooper
Journal:
Biochemical Journal
Biochem J (2006) 399 (3): 513–524.
Published: 13 October 2006
...Jacqueline Dunne; Alexis Caron; Patrick Menu; Abdu I. Alayash; Paul W. Buehler; Michael T. Wilson; Radu Silaghi-Dumitrescu; Beatrice Faivre; Chris E. Cooper Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin...
Abstract
Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 μM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process ( t 1/2 =1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.
Articles
Cinzia Verde, M. Cristina De Rosa, Daniela Giordano, Donato Mosca, Donatella De Pascale, Luca Raiola, Ennio Cocca, Vitale Carratore, Bruno Giardina, Guido Di Prisco
Journal:
Biochemical Journal
Biochem J (2005) 389 (2): 297–306.
Published: 05 July 2005
... the oxygen-transport haem protein from the Antarctic skate Bathyraja eatonii and from the Arctic skate Raja hyperborea (both of the subclass Elasmobranchii, order Rajiformes, family Rajidae) is reported in the present paper. These species have a single major haemoglobin (Hb 1; over 80% of the total...
Abstract
Cartilaginous fish are very ancient organisms. In the Antarctic sea, the modern chondrichthyan genera are poorly represented, with only three species of sharks and eight species of skates; the paucity of chondrichthyans is probably an ecological consequence of unusual trophic or habitat conditions in the Southern Ocean. In the Arctic, there are 26 species belonging to the class Chondrichthyes. Fish in the two polar regions have been subjected to different regional histories that have influenced the development of diversity: Antarctic marine organisms are highly stenothermal, in response to stable water temperatures, whereas the Arctic communities are exposed to seasonal temperature variations. The structure and function of the oxygen-transport haem protein from the Antarctic skate Bathyraja eatonii and from the Arctic skate Raja hyperborea (both of the subclass Elasmobranchii, order Rajiformes, family Rajidae) is reported in the present paper. These species have a single major haemoglobin (Hb 1; over 80% of the total). The Bohr-proton and the organophosphate-binding sites are absent. Thus the haemoglobins of northern and southern polar skates appear functionally similar, whereas differences were observed with several temperate elasmobranchs. Such evidence suggests that, in temperate and polar habitats, physiological adaptations have evolved along distinct pathways, whereas, in this case, the effect of the differences characterizing the two polar environments is negligible.
Articles
Journal:
Biochemical Journal
Biochem J (2005) 385 (2): 409–418.
Published: 07 January 2005
... ). 9 7 2004 26 8 2004 10 9 2004 10 9 2004 The Biochemical Society, London 2005 artemisinin haemoglobin haemozoin Plasmodium falciparum histidine-rich protein II (PfHRP II) redox activity Malaria, one of the commonest diseases in tropical countries, is...
Abstract
Elucidation of the principal targets of the action of the antimalarial drug artemisinin is an ongoing pursuit that is important for understanding the action of this drug and for the development of more potent analogues. We have examined the chemical reaction of Hb with artemisinin. The protein-bound haem in Hb has been found to react with artemisinin much faster than is the case with free haem. It appears that the uptake of Hb and the accumulation of artemisinin into the food vacuole, together with the preferred reactivity of artemisinin with haem in Hb, may make Hb the primary target of artemisinin's antimalarial action. Both monoalkylated (HA) and dialkylated (HAA) haem derivatives of artemisinin have been isolated. These ‘haemarts’ bind to PfHRP II ( Plasmodium falciparum histidine-rich protein II), inhibiting haemozoin formation, and possess a significantly decreased ability to oxidize ascorbic acid. The accelerated formation of HAA from Hb is expected to decrease the ratio of haem to its alkylated derivatives. The haemarts that are generated from ‘haemartoglobins’ may bring about the death of malaria parasite by a two-pronged effect of stalling the formation of haemozoin by the competitive inhibition of haem binding to its templates and creating a more reducing environment that is not conducive to the formation of haemozoin.
Articles
Journal:
Biochemical Journal
Biochem J (2004) 384 (2): 367–375.
Published: 23 November 2004
...Yiping JIA; Somasundaram RAMASAMY; Francine WOOD; Abdu I. ALAYASH; Joseph M. RIFKIND O -R-polyHbA 0 is an intra- and intermolecularly O -raffinose cross-linked derivative of deoxygenated human haemoglobin developed as an oxygen therapeutic. When compared with its native protein (HbA 0 ), O -R...
Abstract
O -R-polyHbA 0 is an intra- and intermolecularly O -raffinose cross-linked derivative of deoxygenated human haemoglobin developed as an oxygen therapeutic. When compared with its native protein (HbA 0 ), O -R-polyHbA 0 was found to be locked in the T (tense) quaternary conformation with a lower oxygen affinity, a reduced Bohr effect (50% of HbA 0 ) and no measurable cooperativity ( h =1). The kinetics of oxygen and CO binding to the protein indicate lower ‘on’ rates and faster ‘off’ rates than HbA 0 and the absence of effects of inositol hexaphosphate (IHP) on the kinetics. Other properties consistent with a T-like conformation are inaccessibility of the βCys-93 thiol group of O -R-polyHbA 0 , the hyperfine splitting from nitrogen in the EPR spectrum of the Fe(II)NO complex of O -R-polyHbA 0 and decreased flexibility in the distal haem pocket, as indicated by low-spin bis-histidine complexes detected by EPR of oxidized chains. A comparison of the properties of O -R-polyHbA 0 with those of HbA 0 with and without IHP, as well as the reaction of nitrite with deoxygenated haemoglobin, provide additional insights into the variations in the conformation of T-state haemoglobin in solution (modifications of the T state produced by adding organic phosphates, like IHP and 2,3-diphosphoglycerate). Although the physiological ramifications of locking HbA 0 in the T conformation with the O -raffinose are still unknown, valuable insights into haemoglobin function are provided by these studies of O -R-polyHbA 0 .
Articles
Kim D. VANDEGRIFF, Andrea BELLELLI, Michele SAMAJA, Ashok MALAVALLI, Maurizio BRUNORI, Robert M. WINSLOW
Journal:
Biochemical Journal
Biochem J (2004) 382 (1): 183–189.
Published: 10 August 2004
...Kim D. VANDEGRIFF; Andrea BELLELLI; Michele SAMAJA; Ashok MALAVALLI; Maurizio BRUNORI; Robert M. WINSLOW The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin...
Abstract
The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin, MalPEG-Hb [maleimide-activated PEG-conjugated haemoglobin], is non-hypertensive with unique physicochemical properties: high O 2 affinity, low co-operativity and large molecular radius. It is therefore of interest to compare the ligand-binding properties of MalPEG-Hb with unmodified cell-free HbA (stroma-free human haemoglobin). NO association rates for deoxy and oxyMalPEG-Hb and HbA were found to be identical. These results confirm the lack of correlation between hypertension and NO for a similar modified haemoglobin with high molecular radius and low p 50 ( p O 2 at which haemoglobin is half-saturated with O 2 ) [Rohlfs, Bruner, Chiu, Gonzales, Gonzales, Magde, Magde, Vandegriff and Winslow (1998) J. Biol. Chem. 273 , 12128–12134]. The R-state O 2 association kinetic constants were also the same for the two haemoglobins. However, even though the p 50 of MalPEG-Hb is approx. half of that of HbA, the biphasic O 2 dissociation rates measured at relatively high p O 2 (150 Torr) were 2-fold higher, giving rise to a 2-fold lower R-state equilibrium association constant for MalPEG-Hb compared with HbA. Thus the O 2 affinity of MalPEG-Hb is higher only at p O 2 values lower than the intersection point of the O 2 equilibrium curves for MalPEG-Hb and HbA. In summary, the present studies found similar rates of NO binding to HbA and MalPEG-Hb, eliminating the possibility that the lack of vasoactivity of MalPEG-Hb is simply the result of reduced molecular reactivity with NO. Alternatively, the unique O 2 -binding characteristics with low p 50 and co-operativity suggest that the ‘R-state’ conformation of MalPEG-Hb is in a more T-state configuration and restricted from conformational change.
Articles
Journal:
Biochemical Journal
Biochem J (2004) 380 (3): 889–896.
Published: 15 June 2004
...M. Cristina DE ROSA; Massimo CASTAGNOLA; Claudia BERTONATI; Antonio GALTIERI; Bruno GIARDINA Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower Δ H of oxygenation compared with the majority of mammalian haemoglobins...
Abstract
Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower Δ H of oxygenation compared with the majority of mammalian haemoglobins from temperate species (exceptions are represented by some cold-resistant species, such as cow, horse and pig). This has been interpreted as an adaptive mechanism of great importance from a physiological point of view. To date, the molecular basis of this thermodynamic characteristic is still not known. In the present study, we show that binding of extra chloride (with respect to adult human Hb) ions to Hb would significantly contribute to lowering the overall heat of oxygenation, thus providing a molecular basis for the low effect of temperature on the oxygenation–deoxygenation cycle. To this aim, the oxygen binding properties of bovine Hb, bear ( Ursus arctos ) Hb and horse Hb, which are representative of this series of haemoglobins, have been studied with special regard to the effect of heterotropic ligands, such as organic phosphates (namely 2,3-diphosphoglycerate) and chloride. Functional results are consistent with a mechanism for ligand binding that involves an additional binding site for chloride ion. Analysis of computational chemistry results, obtained by the GRID program, further confirm the hypothesis that the reason for the lower Δ H of oxygenation is mainly due to an increase in the number of the oxygen-linked chloride-binding sites.
Articles
Journal:
Biochemical Journal
Biochem J (2004) 379 (3): 833–840.
Published: 01 May 2004
... generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin, under conditions where Lys-X protease activity was unaffected, prevented the production of µ-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These...
Abstract
The black pigment of Porphyromonas gingivalis is composed of the µ-oxo bishaem complex of Fe(III) protoporphyrin IX (µ-oxo oligomer, dimeric haem), namely [Fe(III)PPIX] 2 O. P. gingivalis W50 and Rgp (Arg-gingipain)- and Kgp (Lys-gingipain)-deficient mutants K1A, D7, E8 and W501 [Aduse-Opoku, Davies, Gallagher, Hashim, Evans, Rangarajan, Slaney and Curtis (2000) Microbiology 146 , 1933–1940] were grown on horse blood/agar for 14 days and examined for the production of µ-oxo bishaem. µ-oxo Bishaem was detected by UV–visible, Mössbauer and Raman spectroscopies in wild-type W50 and in the black-pigmented RgpA- and RgpB-deficient mutants (W501 and D7 respectively), whereas no haem species were detected in the straw-coloured colonies of Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of µ-oxo bishaem, but of a high-spin monomeric Fe(III) protoporphyrin IX species (possibly a haem–albumin complex). In vitro incubation of oxyhaemoglobin with cells of the W50 strain and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in the formation of µ-oxo bishaem via methaemoglobin as an intermediate. Although the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into µ-oxo bishaem. The double RgpA/RgpB knockout was also not capable of producing µ-oxo bishaem from oxyhaemoglobin, but instead generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin, under conditions where Lys-X protease activity was unaffected, prevented the production of µ-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These results show that one or both of RgpA and RgpB gingipains, in addition to the lysine-specific gingipain, is necessary for the production of µ-oxo bishaem from haemoglobin by whole cells of P. gingivalis .
Articles
Byoung-Kuk NA, Bhaskar R. SHENAI, Puran S. SIJWALI, Youngchool CHOE, Kailash C. PANDEY, Ajay SINGH, Charles S. CRAIK, Philip J. ROSENTHAL
Journal:
Biochemical Journal
Biochem J (2004) 378 (2): 529–538.
Published: 01 March 2004
...Byoung-Kuk NA; Bhaskar R. SHENAI; Puran S. SIJWALI; Youngchool CHOE; Kailash C. PANDEY; Ajay SINGH; Charles S. CRAIK; Philip J. ROSENTHAL Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in...
Abstract
Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum , suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax , the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli . The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.
Articles
Anna L. P. CHAPMAN, Christine C. WINTERBOURN, Stephen O. BRENNAN, T. William JORDAN, Anthony J. KETTLE
Journal:
Biochemical Journal
Biochem J (2003) 375 (1): 33–40.
Published: 01 October 2003
... inflammatory tissue injury. 1 To whom correspondence should be addressed (e-mail christine.winterbourn@chmeds.ac.nz ). 9 5 2003 30 6 2003 9 7 2003 9 7 2003 The Biochemical Society, London ©2003 2003 haemoglobin myeloperoxidase myoglobin neutrophil protein aggregation...
Abstract
Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1–20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.
Articles
Mariagiuseppina PELLEGRINI, Bruno GIARDINA, Cinzia VERDE, Vito CARRATORE, Alessandra OLIANAS, Luigi SOLLAI, Maria T. SANNA, Massimo CASTAGNOLA, Guido di PRISCO
Journal:
Biochemical Journal
Biochem J (2003) 372 (3): 679–686.
Published: 15 June 2003
...Mariagiuseppina PELLEGRINI; Bruno GIARDINA; Cinzia VERDE; Vito CARRATORE; Alessandra OLIANAS; Luigi SOLLAI; Maria T. SANNA; Massimo CASTAGNOLA; Guido di PRISCO The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing...
Abstract
The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the α- and β-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-α(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.
Articles
Ajay SINGH, Bhaskar R. SHENAI, Youngchool CHOE, Jiri GUT, Puran S. SIJWALI, Charles S. CRAIK, Philip J. ROSENTHAL
Journal:
Biochemical Journal
Biochem J (2002) 368 (1): 273–281.
Published: 15 November 2002
... with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala 23 with Gly...
Abstract
Cysteine proteases of Plasmodium falciparum , known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly 23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli , solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala 23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity ( k cat / K m increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.
Articles
Journal:
Biochemical Journal
Biochem J (2002) 365 (3): 801–808.
Published: 01 August 2002
... removing fructosamine residues from proteins. In the present study, we show that incubation of human erythrocytes with 200mM glucose not only caused the progressive formation of glycated haemoglobin, but also increased the level of an anionic form of haemoglobin containing alkali-labile phosphate, to...
Abstract
Fructosamine 3-kinase, which phosphorylates low-molecular-mass and protein-bound fructosamines on the third carbon of their deoxyfructose moiety, is quite active in erythrocytes, and was proposed to initiate a process removing fructosamine residues from proteins. In the present study, we show that incubation of human erythrocytes with 200mM glucose not only caused the progressive formation of glycated haemoglobin, but also increased the level of an anionic form of haemoglobin containing alkali-labile phosphate, to approx. 5% of total haemoglobin. 1-Deoxy-1-morpholinofructose (DMF), a substrate and competitive inhibitor of fructosamine 3-kinase, doubled the rate of accumulation of glycated haemoglobin, but markedly decreased the amount of haemoglobin containing alkali-labile phosphate. The latter corresponds therefore to haemoglobin bound to a fructosamine 3-phosphate group (FN3P-Hb). Returning erythrocytes incubated with 200mM glucose and DMF to a low-glucose medium devoid of DMF caused a decrease in the amount of glycated haemoglobin, a transient increase in FN3P-Hb and a net decrease in the sum (glycated haemoglobin+FN3P-Hb). These effects were prevented by DMF, indicating that fructosamine 3-kinase is involved in the removal of fructosamine residues. The second step of this ‘deglycation’ process is most likely a spontaneous decomposition of the fructosamine 3-phosphate residues to a free amine, 3-deoxyglucosone and P i . This is consistent with the findings that 2-oxo-3-deoxygluconate, the product of 3-deoxyglucosone oxidation, is formed in erythrocytes incubated for 2days with 200mM glucose in a sufficient amount to account for the removal of fructosamine residues from proteins, and that DMF appears to inhibit the formation of 2-oxo-3-deoxygluconate from elevated glucose concentrations.
Articles
Journal:
Biochemical Journal
Biochem J (2001) 360 (2): 481–489.
Published: 26 November 2001
...Puran S. SIJWALI; Bhaskar R. SHENAI; Jiri GUT; Ajay SINGH; Philip J. ROSENTHAL In the malaria parasite Plasmodium falciparum , erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis...
Abstract
In the malaria parasite Plasmodium falciparum , erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P 2 position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.
Articles
Amit V. PANDEY, Himani BISHT, Vinod K. BABBARWAL, Jaya SRIVASTAVA, Kailash C. PANDEY, Virander S. CHAUHAN
Journal:
Biochemical Journal
Biochem J (2001) 355 (2): 333–338.
Published: 06 April 2001
...Amit V. PANDEY; Himani BISHT; Vinod K. BABBARWAL; Jaya SRIVASTAVA; Kailash C. PANDEY; Virander S. CHAUHAN The haem detoxification pathway of the malaria parasite Plasmodium falciparum is a potential biochemical target for drug development. Free haem, released after haemoglobin degradation, is...
Abstract
The haem detoxification pathway of the malaria parasite Plasmodium falciparum is a potential biochemical target for drug development. Free haem, released after haemoglobin degradation, is polymerized by the parasite to form haemozoin pigment. Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2) has been implicated as the catalytic scaffold for detoxification of haem in the malaria parasite. Previously we have shown that a hexapeptide repeat sequence (Ala-His-His-Ala-Ala-Asp), which appears 33 times in Pfhrp-2, may be the major haem binding site in this protein. The haem binding studies carried out by ourselves indicate that up to 18 equivalents of haem could be bound by this protein with an observed K d of 0.94 µ M. Absorbance spectroscopy provides evidence that chloroquine is capable of extracting haem bound to Pfhrp-2. This was supported by the K d value, of 37nM, observed for the haem-chloroquine complex. The native PAGE studies reveal that the formation of the haem-Pfhrp-2 complex is disrupted by chloroquine. These results indicate that chloroquine may be acting by inhibiting haem detoxification/binding to Pfhrp-2. Moreover, the higher affinity of chloroquine for haem than Pfhrp-2 suggests a possible mechanism of action for chloroquine; it may remove the haem bound to Pfhrp-2 and form a complex that is toxic to the parasite.
Articles
Journal:
Biochemical Journal
Biochem J (2001) 353 (2): 207–213.
Published: 08 January 2001
... The Biochemical Society, London © 2001 2001 bacterial NO detoxification haemoglobin NO oxygenase Biochem. J. (2001) 353, 207 213 (Printed in Great Britain) 207 Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the...
Abstract
A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H 2 O 2 accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H 2 O 2 probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean K m for oxygen was 80 µ M; the addition of 15 µ M FAD decreased the K m for oxygen to 15 µ M without a change in V max but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O 2 . Purified Hmp consumed NO in the absence of added FAD (approx. 1 O 2 per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O 2 consumption required approx. 47 µ M O 2 ; NO removal was ineffective at physiologically relevant O 2 concentrations (below approx. 30 µ M O 2 ). On exhaustion of O 2 , NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1% of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.
Articles
Journal:
Biochemical Journal
Biochem J (1999) 343 (3): 681–685.
Published: 25 October 1999
...Tao ZHENG; Thomas BRITTAIN; Nicholas J. WATMOUGH; Roy E. WEBER The role of the amino acid at position α 38 in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position α 38 (which is totally conserved in all mammals) is changed to a Gln, the equilibrium...
Abstract
The role of the amino acid at position α 38 in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position α 38 (which is totally conserved in all mammals) is changed to a Gln, the equilibrium properties of the protein are significantly altered. Equilibrium and kinetic data show that the R-state properties of the protein are essentially unaffected by the mutation whilst the allosteric equilibrium and T-state properties are changed. Mutation of the naturally occurring Gln 38 of the human embryonic haemoglobin ζ-chain (the only known non-Thr containing globin) to a Thr residue shows the converse change in properties produced by the adult mutation, although in this case the situation is complicated by significant chain heterogeneity in the T state. An extension of the two-state model of co-operativity is presented to describe quantitatively the equilibrium ligand binding in the presence of T-state chain heterogeneity. A molecular model is described in which the putative interaction of αGln 38 and βTyr 145 is identified which make a significant contribution to the previously reported unusual ligand-binding properties of the ζ-chain containing human embryonic haemoglobins.