RESUMO
Essentials Patients with hemophilia A and inhibitors receiving emicizumab experience breakthrough bleeding. Safety concerns may exist when combining emicizumab with bypassing agents. Combined bypassing agent and bispecific antibody increased thrombin generation up to 17-fold. Thrombotic effects should be considered when combining emicizumab with plasma bypassing agent. SUMMARY: Background Investigational non-factor products such as emicizumab offer a treatment option for patients with hemophilia and inhibitors. However, their mechanism of action raises questions regarding safety when they are combined with treatments for breakthrough bleeding. Objectives To evaluate in vitro thrombin generation (TG) and clot formation for combinations of activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and a sequence-identical analog of emicizumab (SIA). Methods Therapeutic concentrations of SIA (20-600 nm) alone or with aPCC (0.05-1 U mL-1 ), isolated aPCC components or rFVIIa (0.88-5.25 µg mL-1 ) were tested for TG and compared with reference ranges for healthy donor plasma. Coagulation of FVIII-inhibited blood was determined with a widely established method, i.e. rotational thromboelastometry (ROTEM), and confirmed with the Total Thrombus-formation Analysis System. Results and conclusions SIA (600 nm) or aPCC (0.5 U mL-1 ) alone resulted in peak thrombin levels of 21.4 nm and 38.6 nm, respectively, both of which are lower than normal (83.7 ± 29.8 nm). SIA plus aPCC (0.5 U mL-1 ) increased the peak thrombin level 17-fold over SIA alone, exceeding the reference plasma value by 4.2-fold. This hypercoagulable effect occurred with 600 nmSIA combined with as little as 0.25 U mL-1 aPCC, confirmed by ROTEM. FIX was the main driver for enhanced TG. SIA plus rFVIIa (1.75 µg mL-1 ) induced a 1.8-fold increase in the peak thrombin level in platelet-rich plasma, but it did not reach the normal range. These in vitro experiments demonstrate excessive TG after administration of a combination of aPCC and SIA at clinically relevant doses. Careful judgement may be required when breakthrough bleeding is treated in patients receiving emicizumab.
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BACKGROUND: Tissue factor pathway inhibitor-α (TFPIα) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPIα and FXa, which shuts off the initiation of coagulation via the extrinsic pathway. AIM: To investigate whether direct inhibition of FXa by TFPIα independently of TF plays a role in downregulating coagulation. METHODS: Inhibition of FXa by TFPIα in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPIα and its cofactor, protein S, were quantified: (i) after neutralization of TFPIα and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPIα or protein S. RESULTS: Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPIα requires the presence of protein S. TFPIα and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPIα, which was completely dependent on the presence of protein S. CONCLUSION: Direct inhibition of FXa by TFPIα contributes to the downregulation of coagulation.
Assuntos
Coagulação Sanguínea , Lipoproteínas/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismo , Testes de Coagulação Sanguínea , Regulação para Baixo , Fator IXa/metabolismo , Fator Xa/metabolismo , Humanos , Cinética , Proteína S/metabolismoRESUMO
BACKGROUND: TFPI is a Kunitz-type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVIIa. All three Kunitz domains (KD1, KD2, and KD3) and protein S are required for optimal inhibition of FXa and FVIIa. There is limited information on Kunitz domain requirements of the inhibition of TF:FVIIa-catalyzed FIX and FX activation by TFPI. AIM: To investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation. METHODS: Inhibition of TF:FVIIa-catalyzed FX and FIX activation by full-length TFPI (TFPIFL ) and TFPI constructs was quantified from progress curves of FXa and FIXa generation measured with chromogenic substrates. RESULTS AND CONCLUSIONS: TFPIFL inhibited TF:FVIIa-catalyzed FIX activation with a Ki of 16.7 nmol L(-1) . Protein S reduced the Ki to 1.0 nmol L(-1) . TFPI1-150 and KD1-KD2 had 10-fold higher Ki values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa-catalyzed FIX activation (Ki >800 nm). FX activation was measured at limiting FVIIa and excess TF or vice versa. At both conditions, TFPIFL , TFPI1-150 , and KD1-KD2 showed similar inhibition of FX activation. However, at low phospholipid concentrations, TFPIFL was ~ 15-fold more active than TFPI1-150 or KD1-KD2. Apparently, excess phospholipids act as a kind of sink for TFPIFL , limiting its availability for TF:FVIIa inhibition. Preformed FXa:TFPIFL/1-150 complexes rapidly and stoichiometrically inhibited FIX and FX activation by TF:FVIIa, indicating that binary TFPI:FXa complex formation is the limiting step in TF:FVIIa inhibition. Protein S also enhanced inhibition of TF:FVIIa-catalyzed FX activation by TFPI.
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Coagulação Sanguínea , Fator IXa/metabolismo , Fator VIIa/metabolismo , Inibidores do Fator Xa/metabolismo , Fator Xa/metabolismo , Lipoproteínas/metabolismo , Tromboplastina/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Catálise , Relação Dose-Resposta a Droga , Fator VIIa/antagonistas & inibidores , Inibidores do Fator Xa/farmacologia , Humanos , Cinética , Lipoproteínas/farmacologia , Fosfolipídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína S/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Fucoidan is a highly complex sulfated polysaccharide commonly extracted from brown seaweed. In addition to their many biological activities, fucoidans have recently been demonstrated to inhibit or increase coagulation at different concentration ranges. Their structural features, i.e. molecular weight (Mw), Mw distribution, degree of sulfation, monosaccharide composition, and different linkages, are known to affect these activities. Therefore, structure-activity relationship (SAR) analysis of fucoidan is crucial for its potential use as a procoagulant. In this study, Fucus vesiculosus (F.v.) fucoidan was fractionated by charge and size as well as over- and desulfated to different degrees to yield preparations with various structural properties. The fractions' pro- and anticoagulant activities were assessed by calibrated automated thrombography (CAT) and activated partial thromboplastin time(aPTT) assays. Binding to and inhibition of the anticoagulant protein tissue factor pathway inhibitor (TFPI) and the ability to activate coagulation via the contact pathway were also investigated. This paper discusses the impact of charge density, size, and sugar composition on fucoidan's pro- and anticoagulant activities. Fucoidan requires a minimal charge density of 0.5 sulfates per sugar unit and a size of 70 sugar units to demonstrate desired procoagulant activities for improvement of haemostasis in factor VIII/factor IX-deficient plasma.
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Anticoagulantes/uso terapêutico , Coagulantes/uso terapêutico , Hemofilia A/terapia , Hemofilia B/terapia , Polissacarídeos/uso terapêutico , Fracionamento Químico , Fator IX/genética , Fator VIII/genética , Fucus , Hemofilia A/genética , Hemofilia B/genética , Hemostasia , Humanos , Lipoproteínas/metabolismo , Estrutura Molecular , Peso Molecular , Tempo de Tromboplastina Parcial , Extratos Vegetais/química , Polissacarídeos/química , Ligação Proteica , Relação Estrutura-Atividade , Ésteres do Ácido Sulfúrico/químicaRESUMO
UNLABELLED: Treatment of haemophilia has vastly improved over the last years, but many needs are still unmet. Baxter is continuously pursuing the aim to provide new therapeutic options to patients with haemophilia and to their treating physicians. In fact, there are several opportunities to improve existing therapies, e.g., by new indications for existing products, the introduction of new products, and by novel therapeutic approaches other than factor replacement. Among these, Baxter is working on a number of innovations, such as pharmacokinetics-tailored factor VIII prophylaxis, bypassing agent prophylaxis with FEIBA in inhibitor patients, development of a longer acting pegylated recombinant FVIII, a new recombinant factor IX, a new recombinant factor FVIIa, the first recombinant von Willebrand factor, recombinant ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) as well as gene therapy to cure haemophilia B. CONCLUSION: Baxter is truly committed to the benefit for the patient, and therefore engaged in providing a more and more individualized treatment, in increasing efficiency of current products, in developing new products and new approaches with added value.
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Proteínas ADAM/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Fatores de Coagulação Sanguínea/uso terapêutico , Desenho de Fármacos , Terapia Genética/métodos , Hemofilia A/tratamento farmacológico , Hemofilia A/prevenção & controle , Proteína ADAMTS13 , Humanos , Proteínas Recombinantes/uso terapêuticoRESUMO
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a multi-Kunitz domain protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. OBJECTIVES: To investigate the role of the three Kunitz domains (KDs) of TFPI in FVIIa inhibition using full-length TFPI (TFPIfl ) and truncated TFPI constructs. METHODS: Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPIfl /TFPI constructs was quantified with a FVIIa-specific chromogenic substrate. RESULTS AND CONCLUSIONS: TFPIfl inhibited TF-FVIIa via a monophasic reaction, which is rather slow at low TFPIfl concentrations (t½ ≈ 5 min at 2 nm TFPI) and has a Ki of 4.6 nm. In the presence of sTF and without TF, TFPIfl was a poor FVIIa inhibitor, with Ki values of 122 nm and 1118 nm, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPIfl . TFPI constructs without the KD3-c-terminus (TFPI1-150 and KD1-KD2) were 7-10-fold less effective than TFPIfl in inhibiting TF-FVIIa and sTF-FVIIa, indicating that the KD3-C-terminus significantly contributes to direct inhibition of FVIIa by TFPI. Compared with KD1-KD2, KD1 was a poor TF-FVIIa inhibitor (Ki =434 nm), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPIfl (Ki =0.7 nm). In the presence of FXa, a tight quaternary TF-FVIIa-TFPI-FXa complex is formed with TFPIfl , TFPI1-150 and KD1-KD2, with Ki values of < 0.15 nm, 0.5 nm and 0.8 nm, respectively, indicating the KD3-C-terminus is not a prerequisite for quaternary complex formation. Phospholipids and the Gla-domain of FXa are required for quaternary complex formation.
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Fator VIIa/antagonistas & inibidores , Lipoproteínas/farmacologia , Sequência de Aminoácidos , Humanos , Lipoproteínas/química , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the major inhibitor of tissue factor-initiated coagulation, making it an interesting and novel therapeutic target in hemophilia treatment. The aptamer BAX499 (formerly ARC19499) is designed to improve hemostasis by specifically inhibiting TFPI. OBJECTIVES: The aim of the study was to examine the concentration-dependent augmentation of clotting by BAX499. METHODS: Whole blood clot formation was quantified by rotational thromboelastometry and thromboelastography, and thrombin generation in platelet-poor plasma was assessed with the calibrated automated thrombogram, in samples from patients with congenital hemophilia A (N=55) and B (N=11), patients with acquired hemophilia A (N=1), and healthy controls (N=37). RESULTS: BAX499 significantly improved clotting of samples from hemophilic patients in a concentration-dependent manner, resulting in clotting profiles in samples from patients with severe hemophilia that were similar to those of healthy controls. CONCLUSION: BAX499 improved ex vivo clotting parameters in blood and plasma from patients with hemophilia A and B with different severity of disease, and also in a patient with acquired hemophilia. These results further support the contention that anti TFPI strategies may be an effective treatment for hemophilic patients.
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Aptâmeros de Nucleotídeos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Hemofilia A/sangue , Hemofilia B/sangue , Hemostáticos/farmacologia , Lipoproteínas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Adolescente , Adulto , Idoso , Áustria , Coagulação Sanguínea/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Hemofilia A/diagnóstico , Hemofilia A/genética , Hemofilia B/diagnóstico , Hemofilia B/genética , Humanos , Índia , Lipoproteínas/sangue , Lipoproteínas/genética , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Tromboelastografia , Trombina/metabolismo , Tempo de Coagulação do Sangue Total , Adulto JovemRESUMO
BACKGROUND: Activated coagulation factor IX (FIXa) has low catalytic activity towards its physiologic substrate FX when activated FVIII (FVIIIa) is absent. One reason for this is that the FIX surface loop 99 stabilizes FIXa in a conformation that limits access of FX to the active site. OBJECTIVES: To investigate the effect of mutations in loop 99 and in the active site on FIXa activity with and without FVIIIa. METHODS: Five full-length FIX mutants with amino acid exchanges in the catalytic domain of FIX were constructed and characterized by measuring their activity in FX activation in model systems and in plasma. RESULTS AND CONCLUSIONS: The mutants showed no or marginally improved catalytic properties in FX activation by the intrinsic tenase complex (FIXa-FVIIIa-Ca(2+)-phospholipid). The combination of mutations Y94F and K98T hardly affected FX activation in the presence of FVIIIa, but yielded a FIX molecule that, in FIX-depleted plasma, had approximately 2.5-fold higher clotting activity and approximately 3.5-fold higher activity in a thrombin generation assay than plasma-derived FIX (pdFIX). Two FIXa mutants had considerably increased activities towards FX in the absence of FVIIIa. FIXa-Y94F/K98T/Y177F/I213V/E219G (FIXa-L) and FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIXa-M) activated FX with catalytic efficiencies (k(cat)/K(m)) that, as compared with activated pdFIX, were increased 17-fold and six-fold, respectively. However, in plasma, their zymogen forms performed similarly to pdFIX. This indicates that the introduced mutations not only affected the activity of FIXa but may have also influenced the lifetime of the activated mutant molecules in plasma by modifying their activation and/or inhibition rates.
Assuntos
Fator IX/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Catálise , Domínio Catalítico , Compostos Cromogênicos/metabolismo , Precursores Enzimáticos/metabolismo , Fator IX/química , Fator IX/metabolismo , Fator VIII/metabolismo , Fator X/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Oligopeptídeos/metabolismo , Tempo de Tromboplastina Parcial , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Trombina/biossínteseRESUMO
BACKGROUND: Factor VIIIa (FVIIIa) binds to activated FIX and enhances the activation of FX by several orders of magnitude. Deficiency of FVIII causes the bleeding disorder hemophilia A and is treated by i.v. infusion of FVIII concentrates. OBJECTIVES: To explore whether or not FVIII activity can be supplied by alternative molecules, e.g. molecules with FIXa-binding activity. METHODS: Conventional hybdridoma technology was used to discover antibodies exhibiting FVIII-like activity. RESULTS: We identified a series of antibodies specific for human FIX that mimicked the stimulatory effect of FVIIIa on FIXa. Upon binding to human FIXa, these antibodies enhanced the protease activity of FIXa towards its natural substrate FX about tenfold. A similar enhancement was also achieved with 5 pm FVIIIa (i.e. 16 mU mL(-1) or 1.6% activated FVIII). Procoagulant activity of these anti-FIXa antibodies was observed in model systems containing purified proteins as well as in plasma. CONCLUSION: Our findings show that FVIII can, at least partially, be replaced by an unrelated molecule. Procoagulant antibodies might potentially aid the development of an FVIII substitute for hemophilia A treatment.
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Anticorpos Monoclonais/farmacologia , Fator IX/imunologia , Fator IXa/agonistas , Animais , Anticorpos Monoclonais/imunologia , Sistema Livre de Células , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Fator IXa/imunologia , Fator VIIIa/fisiologia , Fator X/metabolismo , Hemofilia A/tratamento farmacológico , Humanos , Hibridomas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
INTRODUCTION: ADAMTS-13 is a member of A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, primarily synthesized in hepatic stellate cells (HSCs), one of the major cell types transdifferentiating into myofibroblasts during liver fibrosis. However, the association between ADAMTS-13 expression and HSC activation or liver fibrosis is not known. METHODS: In this study, we determined the ADAMTS-13 mRNA, protein, and activity in isolated primary HSCs upon activation on a plastic dish and in liver after administration of carbon tetrachloride (CCl(4)) in rats. RESULTS: We showed that ADAMTS-13 antigen and proteolytic activity in the activated rat HSCs were dramatically increased, whereas ADAMTS-13 mRNA in these cells was only minimally altered. Similarly, the ADAMTS-13 antigen and proteolytic activity in rat liver after CCl(4) injury were also significantly increased, whereas the ADAMTS-13 mRNAs in these liver tissues were only slightly increased compared with normal. Surprisingly, despite the dramatic up-regulation of ADAMTS-13 protein synthesis in the activated HSCs after CCl(4) administration, the plasma levels of ADAMTS-13 protease in rats did not increase concordantly. CONCLUSION: We conclude that the up-regulation of ADAMTS-13 protein expression in rat HSCs during activation in vitro and in vivo suggests the possibility of ADAMTS-13 proteolysis, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries. The data also suggest the minimal contribution of the activated HSCs in regulation of plasma levels of ADAMTS-13 protease.
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Proteínas ADAM/metabolismo , Fígado/metabolismo , Proteína ADAMTS13 , Animais , Western Blotting , Células Cultivadas , Hidrólise , Imuno-Histoquímica , Fígado/citologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N-terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well-preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA-DOM I-II and HSA, followed by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA.
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Albumina Sérica/metabolismo , Varfarina/metabolismo , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Ligantes , Modelos Químicos , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Albumina Sérica/química , Albumina Sérica/genética , Albumina Sérica/isolamento & purificação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Triptofano/químicaRESUMO
Human serum albumin (HSA) is a protein of 66.5 kDa that is composed of three homologous domains, each of which displays specific structural and functional characteristics. HSA is known to undergo different pH-dependent structural transitions, the N-F and F-E transitions in the acid pH region and the N-B transition at slightly alkaline pH. In order to elucidate the structural behavior of the recombinant HSA domains as stand-alone proteins and to investigate the molecular and structural origins of the pH-induced conformational changes of the intact molecule, we have employed fluorescence and circular dichroic methods. Here we provide evidence that the loosening of the HSA structure in the N-F transition takes place primarily in HSA-DOM III and that HSA-DOM I undergoes a structural rearrangement with only minor changes in secondary structure, whereas HSA-DOM II transforms to a molten globule-like state as the pH is reduced. In the pH region of the N-B transition of HSA, HSA-DOM I and HSA-DOM II experience a tertiary structural isomerization, whereas with HSA-DOM III no alterations in tertiary structure are observed, as judged from near-UV CD and fluorescence measurements.
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Conformação Proteica , Albumina Sérica/química , Humanos , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Albumina Sérica/genéticaRESUMO
In an attempt to systematically dissect the ligand binding properties of human serum albumin (HSA), the gene segments encoding each of its three domains were defined based on their conserved homologous structural motifs and separately cloned into a secretion vector for Pichia pastoris. We were able to establish a generally applicable purification protocol based on Cibacron Blue affinity chromatography, suggesting that each of the three domains carries a binding site specific for this ligand. Proteins were characterized by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, gel filtration, N-terminal sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as well as near- and far-UV CD. In addition to the affinity chromatography ligand Cibacron Blue, binding properties toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respectively, were investigated by the measurement of induced circular dichroism. Clear experimental evidence is provided here for the location of the primary hemin binding site to be on domain I of HSA, and for the primary diazepam binding site to be on domain III. Further, secondary binding sites were found for hemin to be located on domains II and III, and for diazepam on domain I. The warfarin binding site was located primarily on domain II, while on domain I, a secondary binding site and/or parts of the primary binding site were found.
Assuntos
Albumina Sérica/química , Sítios de Ligação , Dicroísmo Circular , Corantes , Diazepam/metabolismo , Hemina/metabolismo , Humanos , Ligantes , Modelos Moleculares , Pichia/genética , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Albumina Sérica/genética , Triazinas , Varfarina/metabolismoRESUMO
The Synechocystis PCC 6803 katG gene encodes a dual-functional catalase-peroxidase (EC 1.11.1.7). We have established a system for the high level expression of a fully active recombinant form of this enzyme. Its entire coding DNA was extended using a synthetic oligonucleotide encoding a hexa-histidine tag at the C-terminus and expressed in Escherichia coli [BL21-(DE3)pLysS] using the pET-3a vector. Hemin was added to the culture medium to ensure its proper association with KatG upon induction. The expressed protein was purified to homogeneity by two chromatography steps including a metal chelate affinity and hydrophobic interaction chromatography. The homodimeric acidic protein (pl = 5.4) had a molecular mass of 170 kDa and a Reinheitszahl (A406/A280) of 0.64. The recombinant protein contained high catalase activity (apparent Km = 4.9 +/- 0.25 mM and apparent kcat = 3500 s(-1)) and an appreciable peroxidase activity with o-dianisidine, guaiacol and pyrogallol, but not with NAD(P)H, ferrocytochrome c, ascorbate or glutathione as electron donors. By using both conventional and sequential stopped-flow spectroscopy, formation of compound I with peroxoacetic acid was calculated to be (8.74 +/- 0.26) x 10(3) M(-1) s(-1), whereas compound I reduction by o-dianisidine, pyrogallol and ascorbate was determined to be (2.71 +/- 0.03) x 10(6) M(-1) S(-1), (8.62 +/- 0.21) x 10(4) M(-1) S(-1), and (5.43 +/- 0.19) x 10(3) M(-1) S(-1), respectively. Cyanide binding studies on native and recombinant enzyme indicated that both have the same heme environment. An apparent second-order rate constant for cyanide binding of (4.8 +/- 0.1) x 10(5) M(-1) S(-1) was obtained.