β amylase

The pattern of protein hydrolysis and molecular weight of enzyme-treated chicken foot collagen were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC), respectively. To analyze the pattern of protein hydrolysis, we used the SDS-PAGE protocol described by Laemmli [21 (link)] with 12% separating and 5% stacking gels. Samples were prepared for electrophoresis by boiling in a loading buffer containing 0.1% Coomassie Brilliant Blue R 250 dye (Sigma-Aldrich, USA). Molecular weight distribution and average molar mass were evaluated using an LC-2000 Plus HPLC system (Jasco, Japan). For evaluation, the collagen samples were prepared by filtration through a 0.45-μm membrane using a Shodex Protein KW-802.5 column (I.D. 8 mm × 300 mm; Shodex, Japan). The mobile phase used was 50 mM phosphate buffer containing 0.3 M NaCl at a flow rate of 0.9 ml/min, and the detector recorded absorbance at 220 nm. We used thyroglobulin (669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa), aprotinin (6.5 kDa), and cyanocobalamin (1.3 kDa) as protein standards (all obtained from Sigma-Aldrich) for standard curve construction and evaluation of the average molecular weight of enzyme-hydrolyzed collagen samples.

A Superdex 200 column (10 mm × 300 mm) was used to estimate the apparent molecular mass of purified FtRelA (∼1 mg/ml, ∼13.5 μM). Protein samples were applied (1 ml·min⁻¹) to a pre-equilibrated column in buffer D (50 mM Tris/HCl, pH 7.5, 100 mM KCl). Cytochrome C, carbonic anhydrase, BSA, alcohol dehydrogenase and β-amylase (Sigma–Aldrich) were used as protein standards for calibration of the column. Elution of the protein samples were monitored by absorbance at 280 nm.

Using a fast protein liquid chromatography (GE Healthcare Europe GmbH, Munich, Germany) equipped using a silica gel packed in a TSKgel G2000SWXL column (7.8 mm id × 30 cm), the MM analysis of gelatin was done as previously reported [23 ]. Elution was performed using 0.1 M phosphate buffer; 0.2 M sodium chloride. The standard markers were blue dextran (2000 kDa), apoferritin (443 kDa), β-amylase (200 kDa) and bovine serum albumin (66 kDa) (Sigma-Aldrich). The gelatins and standard markers were loaded into the column at 5 mg/ml. The MM was calculated from the standard curve.

The subunit molecular mass of MpIspS was examined by SDS-PAGE under denaturing conditions, using the proteins of a pre-stained ladder (Bio-Rad) as reference proteins. All protein bands were stained with Coomassie Blue for visualization. The native molecular mass of the enzyme was determined by gel-filtration chromatography on a Superose 12 10/300 GL column (GE Healthcare, Buckinghamshire, UK). The purified enzyme was applied to the column and eluted with 25 mM Tris-HCl (pH 7.4) buffer containing 200 mM NaCl at a flow rate of 1 mL/min. The column was calibrated with apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (13.7 kDa), and carbonic anhydrase (29 kDa) as reference proteins (Sigma-Aldrich), and the native molecular mass of the enzyme was calculated by comparing with the migration length of reference proteins.

In order to analyze the structural properties of NfCPI, electrophoretic analysis of the NfCPI was performed in the presence and absence of SDS or β-mercaptoethanol [β-ME, 5% (vol/vol)] with or without prior heating at 100 °C for 5 min [12 (link),13 (link)]. The native molecular size of NfCPI was also analyzed by gel filtration chromatography using a Superdex 200 HR 10/30 column with an Äcta FPLC system (GE Biosciences, Pittsburgh, PA, USA). The purified NfCPI (1 mg) was loaded onto the column, and the collected fractions (0.5 mL) were analyzed by SDS-PAGE, followed by measurement of their inhibitory activities against NfCB and NfCBL. The column was calibrated with gel filtration size marker proteins (Sigma): blue dextran (2000 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). The K_av value of each size marker protein was calculated using the equation K_av = (V_e − V₀)/(V_t − V₀), where V_e is the elution volume of protein_,V₀ refers to the elution volume of blue dextran, and V_t is the total bed volume.