And SDS-PAGE, respectively. The active and homogenous NOP Receptor/ORL1 Agonist Storage & Stability fractions in

And SDS-PAGE, respectively. The active and homogenous NOP Receptor/ORL1 Agonist Storage & Stability fractions in the cation exchange had been pooled and submitted to 1 cycle of gel filtration on a Sephacryl S200 column preequilibrated with 25 mM Tris-HCL at pH eight.0 Topo II Inhibitor supplier containing 0.six M NaCl. The column was eluted by 100 mM Tris-HCL buffer (pH eight.0) to wash the unbound proteins. The bound proteins have been eluted with linear salt gradients of 1 , two , three , 4 , and 5 NaCl inside the similar buffer. All of the fractions have been analyzed as described above. The active and homogenous fractions were pooled, concentrated, and stored at four C for further evaluation. two.four. Proteolytic Activity Assay. The proteolytic activity of purified protease was measured in accordance with the approach described by Zanphorlin et al. [8] with some modification. The reaction mixture contained 1 mL of 0.five (wv-1 ) azocasein ready in 100 mM Tris-HCl (pH eight.0) buffer and 0.1 mL of enzyme. The mixture was incubated in a water bath at 80 C for 1 h, and 10 (wv-1 ) of 0.3 mL of trichloroacetic acid (TCA) was added to stop the reaction, followed by centrifugation at ten,000 rpm for ten min at room temperature (Microfuge 18 centrifuge, Beckman Coulter, Inc., Krefeld, Germany). The absorbance of the TCA-soluble supernatant was determined at 410 nm utilizing a spectrophotometer (BioMate-3, Thermo Scientific, Alpha Numerix, Woodfield Dr, Webster, NY, USA). A single unit of proteolytic activity is defined as the volume of enzyme causing an increase in absorbance of 0.01. The precise protease activity was expressed as enzyme activity (U) per mg of protein. The handle was run by substituting the enzyme using the identical volume of enzyme extract heated in a boiling water bath for 30 min for inactivation of your enzyme. two.five. Determination of Protein Concentration. Protein concentration was determined by the Bradford [9] system and BSA was made use of as normal. 2.six. Determination of Purity and Molecular Weight of Purified Protease. SDS-PAGE was performed on a minivertical gel electrophoresis unit (Amersham Biosciences) utilizing 15 acrylamide separating gel inside the presence of 0.1 SDS and 4 acrylamide stacking gel containing 0.1 SDS in line with the system described by Laemmli [10]. The SDS minimizing sample buffer and tank buffer have been 0.5 M Tris-HCl (pH six.8) containing 2 SDS and Tris-glycine (0.025 M Tris-HCl, pH eight.three; 0.192 M glycine) in the presence of 0.1 SDS, respectively. Electrophoresis was performed at area temperature, along with the run was conducted at 15 mA and 250 V for the stacking gel and 30 mA and 250 V for the resolving gel. Proteins in2. Material and Methods2.1. Plant Material and Chemicals. Red pitaya fruits (Hylocereus polyrhizus) have been bought from Pasar Borong (Selangor, Malaysia). Ripened pitaya fruits had been selected determined by the size uniformity at the identical stage of ripening absolutely free of visual defects. The fruits had been stored within a cold space at four C until use for the extraction process. All chemical compounds and reagent have been in analytical or electrophoresis grade. SP-Sepharose, Sephacryl S-200, Bradford Reagent, BSA, DTNB, PMSF, EDTA, ovomucoid, iodoacetic acid, bestatin, -mercaptoethanol, PMSF, and trichloroacetic acid (TCA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tris-HCL, Triton X-100, Tween-80, SDS, casein, haemoglobin, acetone, ethanol, isopropanol, and methanol were obtained from Merck (Darmstadt, Germany). 2.two. Extraction of Thermoalkaline Protease. Fresh pitaya fruits (2 Kg) had been cleaned and rinsed thoroughly with sterile distilled water and.