A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUniversidade de São Paulo Faculdade de Medicina Instituto do CoraçãoUniversidade de São Paulo Escola de Educação Física e Esporte Laboratório de Bioquímica da Atividade MotoraUNIFESP, EPM, Depto. de BiofísicaUNIFESP, EPM, Depto. de MedicinaSciEL

Síntese e estudo cinético de novos substratos cromogênicos para algumas serino-proteases

BV UNIFESP: Teses e dissertaçõe

Analysis of secreted protein profile and enzymatic activities from Corynebacterium diphtheriae and Bordetella pertussis on production batch media using peptide quenched fluorescent substrates

Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. the proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. the proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis ( SDS- PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. the proteases were stable and presented enzymatic activity at 378 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.Butantan Inst, Biochem & Biophys Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilButantan Inst, Dept Bacteriol, Sect Aerob Vaccines, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilWeb of Scienc

Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. the whole procedure, including neutrophil purification and kinetic measurements, can be done in 4-5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.Alexander von Humboldt FoundationGerman Research CouncilVaincre la MucoviscidoseFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Proteases & Vectorisat Pulm Fac Med, INSERM, U618, F-37032 Tours, FranceMax Planck Inst Neurobiol, Dept Neuroimmunol, D-82152 Planegg Martinsried, GermanyUniv Tours, F-37032 Tours, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-0404420 São Paulo, BrazilINSERM, U921, F-37032 Tours, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-0404420 São Paulo, BrazilWeb of Scienc

Substrate specificity of kallikrein-related peptidase 13 activated by salts or glycosaminoglycans and a search for natural substrate candidates

KLK13 is a kallikrein-related peptidase preferentially expressed in tonsils, esophagus, testis, salivary glands and cervix. We report the activation of KLK13 by kosmotropic salts and glycosaminoglycans and its substrate specificity by employing a series of five substrates derived from the fluorescence resonance energy transfer (FRET) peptide Abz-KLRSSKQ-EDDnp. KLK13 hydrolyzed all these peptides only at basic residues with highest efficiency for R; furthermore, the S(3) to S(2)' subsites accepted most of the natural amino acids with preference also for basic residues. Using a support-bound FRET peptide library eight peptide substrates were identified containing sequences of proteins found in testis and one with myelin basic protein sequence, each of which was well hydrolyzed by KLK13. Histatins are salivary peptides present in higher primates with broad antifungal and mucosal healing activities that are generated from the hydrolysis from large precursor peptides. KLK13 efficiently hydrolyzed synthetic histatin 3 exclusively at R(25) (DSHAKRHHGYKRKFHEKHHSHRGYR(25)down arrow SNYLYDN) that is the first cleavage observed inside the salivary gland.In conclusion, the observed hydrolytic activities of KLK13 and its co-localization with its activators, glycosaminoglycans in the salivary gland and high concentration of sodium citrate in male reproductive tissues, indicates that KLK13 may play a role in the defense of the upper digestive apparatus and in male reproductive organs. (C) 2011 Elsevier Masson SAS. All rights reserved

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K-i 14 nM).BuXI and BvTI are highly homologous (70%). the major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition.Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. the catalytic efficiency k(cat)/K-m 4.3 x 10(7) M(-1)sec(-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate.Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P-1') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa.Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilEMBRAPA, Campo Grande, MS USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Human tissue kallikreins 3 and 5 can act as plasminogen activator releasing active plasmin

Human tissue kallikreins (KLKs) are a group of serine proteases found in many tissues and biological fluids and are differentially expressed in several specific pathologies. Here, we present evidences of the ability of these enzymes to activate plasminogen. Kallikreins 3 and 5 were able to induce plasmin activity after hydrolyzing plasminogen, and we also verified that plasminogen activation was potentiated in the presence of glycosaminoglycans compared with plasminogen activation by tPA. This finding can shed new light on the plasminogen/plasmin system and its involvement in tumor metastasis, in which kallikreins appear to be upregulated. (C) 2013 Elsevier Inc. All rights reserved