RESUMO
Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.
Assuntos
Papaína/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Ácido Tetratiônico/química , Carica/química , Misturas Complexas/química , Eletroforese em Gel de Poliacrilamida , Látex/química , Peso Molecular , Papaína/química , Proteínas de Plantas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-L: -leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (K (m) = 0.18 ± 0.03 mM, k (cat) = 1.078 ± 0.055 s(-1), k (cat)/K (m) = 5.99 ± 0.57 s(-1) mM(-l)). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman's degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was suggested by molecular modeling.
Assuntos
Apocynaceae/metabolismo , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Látex/química , Sequência de Aminoácidos , Apocynaceae/enzimologia , Apocynaceae/genética , Sequência de Bases , Clonagem Molecular , Cisteína Proteases/genética , Cisteína Proteases/isolamento & purificação , DNA Complementar/genética , Frutas/enzimologia , Frutas/genética , Frutas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , Cinética , Modelos Químicos , Dados de Sequência Molecular , Peso Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders. These pharmacological actions have been attributed to the presence of cysteine proteases in these milky latices. Asclepias curassavica (Asclepiadaceae), "scarlet milkweed" is a perennial subshrub native to South America. In the current paper we report a new approach directed at the selective biochemical and molecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for the proteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases were digested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. No identification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceae latex cysteine proteinases available in databases. From total RNA extracted from latex samples, cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained by cloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequences of 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII, characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain the full-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment and phylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group of papain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMF could be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with very similar physicochemical and functional properties, with advantages over other conventional methods (for instance enzyme kinetics) that are time consuming and afford less reliable results.
Assuntos
Asclepias/enzimologia , Cisteína Endopeptidases/metabolismo , Látex/análise , Clonagem Molecular , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase , Hidrólise , Isoenzimas/genética , Látex/química , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Látex/metabolismo , Sequência de Aminoácidos , Asclepias , Bioquímica/métodos , Caseínas/química , Cátions , Cromatografia por Troca Iônica , Cisteína/química , Relação Dose-Resposta a Droga , Ácido Edético/farmacologia , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Concentração de Íons de Hidrogênio , Íons , Cinética , Látex/química , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Fatores de Tempo , UltracentrifugaçãoRESUMO
A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.
Assuntos
Apocynaceae/enzimologia , Cisteína Endopeptidases/metabolismo , Látex/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Látex/química , Espectrometria de Massas , Fatores de TempoRESUMO
A new cysteine endopeptidase (morrenain b I) has been purified and characterized from the latex of stems and petiols of Morrenia brachystephana Griseb. (Asclepiadaceae). Morrenain b I was the minor proteolytic component in the latex but showed higher specific activity than morrenain b II, which was the main active fraction. Both enzymes showed similar pH profiles and molecular masses, but kinetic parameters and N-terminal sequences were quite distinct, demonstrating that they are different enzymes instead of different forms of the same enzyme.