Luis Juliano

A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.

Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification

Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two‐chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. The low‐molecular‐mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73–82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti‐recombinant fragment C antibody, and the cleavage was in a single bond (Gln‐Phe) with purified and crude TTx. Besides, ELISA applied to the anti‐TTx serum produced at our Institute showed cross‐reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. The results obtained suggest that the use of the new fluorescent substrate, Abz‐synaptobrevin73–82‐EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).

Enzymatic profiling of tetanus and botulinum neurotoxins based on vesicle-associated-membrane protein derived fluorogenic substrates.

Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. In order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed. The assays were conducted with FRET peptides derived from SNAP-25, synaptobrevin and syntaxin. The substrates were cleaved by 2 ng/mL of toxin at different rates (K(cat)/K(M) from 0.028 to 75.9 microM.s(-)) at a single bond, as confirmed by Q-TOF mass spectrometry. Inhibition of the hydrolysis was obtained with EDTA or with specific antibodies directed to each neurotoxin. Different substrate selectivities, especially by BoNT- A and E, suggest that these substrates can be used as a putative method for clostridial toxin quantification and serotype differentiation and could be easily adapted to a high-throughput protocols.

Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.

Met-Lys-Bradykinin-Ser, the kinin released from human kininogen by human pepsin

This study was carried out to show the site in kininogen, using synthetic substrates, that is cleaved by a purified human pepsin component with a molecular weight of 35,000 daltons. The study used 4 (four) intramolecularly quenched fluorogenic substrates containing N- and C-terminal sequences around the methionyl-lysyl-bradykinin (MLBK) region of kininogen: Abz-LMKRP-Eddnp, Abz-MISLMKRP-Eddnp, Abz-FRSSR-Eddnp and Abz-RPPGFSPFRSSRQ-Eddnp. The hydrolysis on N-terminal sequences Abz-LMKRP-Eddnp and Abz-MISLMKRP-EDDnp occurred at L-M linkage and on C-terminal sequences Abz-FRSSR-Eddnp, and Abz-RPPGFSPFRSSRQ-Eddnp occurred at S-S linkage. The released peptide Abz-RPPGFSPFRS was able to contract rat uterus muscle. The results suggest that Met-Lys-Bradykinin-Ser, should be the peptide released from human kininogen by a purified human pepsin component.

Tissue Kallikreins and Related Enzymes: Characterization by Model Oligopeptides

The first purpose of this work was to obtain direct evidence that tissue kallikreins cleave arginyl bonds when the leaving group is Arg-Val, and on the contrary, do not split them when it is Arg-Pro; the second aim was to ascertain whether this specificity could be used as a criterion, for characterizing tissue kallikreins. Two tetrapeptides Ac-Phe-Arg-Arg-Val-NH2 and Ac-Phe-Arg-Arg-Pro-NH2 were synthesized by the solid phase method and purified to homogeneity. They were used as substrates for homogeneous preparations of tissue and plasma kallikreins, as well as for some related serine proteases. Products identification and kinetic analyses were made by HPLC.

TLR4-mediated immunomodulatory properties of the bacterial metalloprotease arazyme in preclinical tumor models

ABSTRACT Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8+ T lymphocytes were the main effector antitumor population, although B and CD4+ T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a promising novel alternative for metastatic melanoma treatment.

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

Abstract 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 β‐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 37°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.