SP-Sephadex equilibrium chromatography of bradykinin and related peptides: Application to trypsin-treated human plasma

An analytical method is deseribed for the separation of bradykinin, Lys-bradykinin, and Met-Lys-bradykinin by equilibrium chromatography on SP-Sephadex C-25 eluted in 0.02 Tris-HCl buffer, pH 8.10, 0.12 NaCl. A second elution buffer, 0.02 Tris-HCl buffer, pH 7.70, 0.06 NaCl, serves as a second parameter for the identification of bradykinin and also separates the hormone from plasma bradykinin-potentiating peptides. Ten to one-hundred nanomoles of each peptide can be recovered in high yields, identified by elution position, and measured by bioassay with the isolated guinea pig ileum. The identification of bradykinin as the peptide released by trypsin acting on acid-denatured plasma is documented as an illustration of the method

Erratum to "Action of plant proteinase inhibitors on enzymes of physiopathological importance" [An Acad Bras Cienc 81(2009): 615-621]

Universidade Federal de São Paulo (UNIFESP) Departamento de BioquímicaUNIFESP, Depto. de BioquímicaSciEL

Preliminary characterization of a Kazal-type serine protease inhibitor from Caiman crocodilus yacare plasma

Blood serine protease inhibitors are becoming better understood and increasingly applied in blood clotting, cancer and other diseases. Reptiles are suitable models for blood coagulation and related processes, moreover, caiman is a good comparative model of a non-poisonous reptile. Recently, we reported the purification of a kininogen, the presence of proteases involved in blood clotting, and a serine protease inhibitor in Caiman crocodilus yacare plasma. in this paper, we described the partial sequence of an inhibitor (CcTI). the inhibitor is an 80-kDa protein, and it inactivates trypsin and chymotrypsin the hydrolysis of specific chromogenic substrates and in the degradation of gelatin. the inhibitor is member of Kazal-type inhibitor family and consists of several domains, its putative reactive: site is Arg-His. (C) 1999 Elsevier Science B.V. All rights reserved.UNIFESP, Escola Paulista Med, Dept Biochem, BR-04044020 São Paulo, BrazilInnenstadt LMU Munchen, Chirurg Klin & Poliklin, Klin Chem & Klin Biochem Abt, D-80336 Munich, GermanySapporo Med Univ, Dept Internal Med 2, Sapporo, Hokkaido, JapanUNIFESP, Escola Paulista Med, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc

A novel subclassification for Kunitz proteinase inhibitors from leguminous seeds

Kunitz-type trypsin inhibitors from legume seeds have been characterized structurally. the presence of Cys-Cys in single or double chains shows a new pattern of proteins structurally not so closely related to STI. Therefore, briefly, with regard to cysteine content, plant Kunitz proteinase inhibitors may be classifed into four groups: no Cys-Cys at all, one, two and more than two Cys residues. Functional properties and diversity of these proteins are also briefly discussed. (C) 2010 Elsevier Masson SAS. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilWeb of Scienc

Heparin modulation of human plasma kallikrein on different substrates and inhibitors

The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein ( huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. the catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12 x 10(4) M-1 S-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40 x 10(5) M-1 S-1 for H-D-ProPhe-Arg-p-nitroanilide, 2.25 x 10(4) M-1 S-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24 x 10(2) M-1 S-1 for factor XII and 5.58 x 10(2) M-1 S-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates ( by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis ( 7.7- and 1.4-fold, respectively). the second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40 x 10(2) M-1 S-1 and 1.70 x 10(4) M-1 S-1, respectively. Heparin improved the inhibition of huPK by these inhibitors ( 3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv São Paulo, Dept Bioquim, Inst Quim, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Interaction of proteinase inhibitors with phospholipid vesicles is modulated by pH

rBbKI and rBbCI, plant recombinant inhibitors from Bauhinia bauhinioides, and BpuTI from Bauhinia purpurea seeds distinctly and specifically block proteolytic enzymes. the secondary structures of those inhibitors were compared and their interactions with phospholipid vesicles were evaluated by the release of calcein and by intrinsic fluorescence of tryptophan residues. the results show that rBbKI, rBbCI and BpuTI are able to interact with phospholipd vesicles and induce membrane permeabilization in a concentration- and pH-dependent manner. the leakage was rapid and extensive at pH 4.5, but at physiological pH, no calcein release was observed. These results may suggest that upon inflammation or microorganism invasion accompanied by lowering of pH, appropriate conditions may occur for the inhibitors to interact with cell membrane and act on specific proteolytic enzyme. (C) 2010 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, BR-85903000 Toledo, PR, BrazilUniv Coimbra, Fac Ciencias & Tecnol, Ctr Neurociencias & Biol Celular, Dept Ciencias Vida, P-3001401 Coimbra, PortugalUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilWeb of Scienc