Bovine serum albumin (BSA), Coomassie Brilliant Blue R-250 (CBB), trichloroacetic acid (TCA), tri (hydroxymethyl) aminomethane (Tris), β-mercaptoethanol, casein, Insulin β chain and Bradford reagent were obtained from Sigma-Aldrich (St. Louis, MO). Acrylamide, Azocasein, Trifluoroacetic acid (TFA) were obtained from Fluka (Milwaukee, WI)
All other chemicals used were of highest purity available from local commercial sources.
Ripe fruit from B. antiacantha was collected from plants grown in most soil with moderate lighting in the Departmento of Rocha (Uruguay) during early autumn 2011. Fruit was stored at -20°C. Plant fruit was deposited in a botanical collection in the Facultad de Química, UdelaR, Montevideo, Uruguay.
The epigyne used were from A. iheringi. Specimens were captured in pitfall traps in the suburb of Marindia (Canelones, Uruguay). The animals were subsequently stored in 70% ethanol at room temperature.
Extraction of proteolytic enzymes
Endocarp of B. antiacantha was first separated from the fruit skin and fiber. The endocarp tissue without seeds was then macerated using a mortar, without extracting medium, while maintaining the tissue and fruit juice cold using an ice bath. Endocarp crude extract (CE) was obtained by centrifugation at 6654 x g (Sigma 3K18, Osterode am Harz, Germany) for 15 min at 4°C and clarification of the supernatant using Wathman filter paper No. 4. CE was fractionated and stored at -20°C. Protein content in CE was determined by the Bradford method (Bradford, 1976) using BSA as standard.
Determination of proteolytic activity
Proteolytic activity was determined using azocasein as substrate by a method modified from Andrew and Asenjo (Andrew BA and Asenjo JA, 1986). Briefly, CE was activated for 15 min at 4°C by addition of β-mercaptoethanol to 15 mM. Then, 340 μL of a 1/200 dilution of activated CE, 340 μL of 1% azocasein solution (0.1 M Tris–HCl buffer, pH 7.2) and 340 μL of 0.1M Tris–HCl, pH 7.2, were mixed and incubated 10 min at 37°C. The reaction was stopped by addition of 340 μL 10% TCA, centrifuged for 30 min at 13226 x g and absorbance at 337 nm was measured. One enzyme unit (EU) is defined as the amount of enzyme required to produce one unit increase in absorbance at 337 nm under conditions tested.
Native PAGE and Zymogram
CE was precipitated at -20°C with addition of four volumes acetone and after centrifugation was suspended in 0.1M Tris–HCl, pH 8.8. 10 μL aliquots were loaded onto 8 × 10 × 0.75 cm 10% polyacrylamide minigels. Two gels were run in parallel at 121 V for 1 h. One gel was fixed and stained with CBB R-250, and the other was placed in contact with an agarose gel of the same dimensions that was. Transfer of proteolytic activity to the agarose gel was detected after drying and staining with CBB R-250 (Westergaar et al., 1980).
A mixture of 1–2 μL crystallization solution (SA or HCCA) and 1 μL of sample were prepared in a 200 μL plastic tube. Volumes of between 0.5-1 μL of this mixture were spotted on MTP 384 target plate polished steel (Bruker Daltonik GmbH) and allowed to evaporate to dryness. Mass spectra were acquired on a Bruker Ultraflex (MALDI-TOF MS) spectrometer equipped with a pulsed nitrogen laser (337 nm), in linear positive ion mode, using a 19 kV acceleration voltage. Molecular mass of protein in CE was determined by MS using SA as matrix.
Proteolytic enzyme specificity
Hydrolysis reactions of reduced insulin β chain were done in 50 mM Tris–HCl, pH 8.0, 20 mM cysteine and 0.0182: 9.1 nmol enzyme- substrate ratio, at 37°C. The reaction was stopped at 0, 5, 15, 90 min and 16 h by adding 8 μL 0.1% TFA (v/v) to 2 μL of reaction mix. The reaction products at these times were crystallized with HCCA matrix in order to be analyzed by MALDI-TOF MS. Validation and positive control digestions were done using papain as reference enzyme, cysteine protease family C1A. The cleavage sites of the proteolytic enzymes in the insulin β chain were determined using GPMAW software v6.0.
Epigyne soft tissue digestion
Epigyne structures were removed from animal specimens and stored in 70% ethanol at room temperature prior to preparation. Samples were washed repeatedly with distilled water before treatment with enzymes. The epigyne, (ca. 10 mg each) were incubated in 1.0 mL diluted enzyme solution at pH 4.0, 6.0 or 8.0, and for different times in a 42°C water bath with continuous stirring. The samples were washed and suspended in water, then sonicated (Sonicador ULTRONICS UL16, Teltow, Germany) for 15 min. Subsequently, they were rinsed again and stored in 70% ethanol at room temperature until microscopically analysis. Negative controls included epigyne incubated without added enzyme in distilled water for 22 h at 42°C followed by sonication and storage. The enzymatic removal was evaluated by optical microscopy.
Samples were prepared for SEM (JOEL 9500, Tokoyo, Japan) using a standard protocol in which the samples were dehydrated in increasing concentrations of ethanol (50, 75 and 100%), then mounted for examination after coating with gold/palladium alloy.