Bovine α chymotrypsin

Egg L-α-phosphatidylcholine (PC, lecithin grade 1, 99% purity) was obtained from Lipid Products (South Nutfield, Surrey, UK). Porcine gastric mucosa pepsin, bovine α-chymotrypsin, pancreatic α-amylase, porcine colipase, porcine pancreatic lipase and bile salts were obtained from Sigma (Poole, Dorset, UK). Lipase for the gastric phase of digestion was a gastric lipase analogue of fungal origin (F-AP15) from Amano Enzyme Inc. (Nagoya, Japan). All flavonoid and other phytochemical standards were obtained from either Sigma-Aldrich (Poole, UK) or Extrasynthese (Genay, France). All solvents were HPLC grade, water was ultra-pure grade, and other chemicals were of AR quality.

The CBG glycoforms were tested for their sensitivity to proteolysis after incubation with
proteases (neutrophil elastase, bovine α-chymotrypsin, LasB) that specifically
target the CBG RCL (Fig. 1C). The amounts of
enzymes used were adjusted to produce ~35–55% reductions in the steroid-binding
capacity of the recombinant un-mutated CBGs. Neutrophil elastase (Elastin Products) was
reconstituted at 0.1 µg/µL in a buffer containing 0.05 M NaAc
(pH 5) and 0.1 M NaCl. Indicated amounts were added to CBG samples in
100 µL 20 mM Tris (pH 8) and incubated for 10 min at
37°C followed by the addition of 5 mM phenylmethanesulfonyl fluoride to stop
reactions, prior to steroid-binding capacity assays or SDS-PAGE. Bovine
α-chymotrypsin (type II from pancreas; Sigma-Aldrich) was reconstituted at
1 µg/µL in 0.1 M Tris–HCl (pH 7.5), 0.5 M NaCl.
Indicated amounts were incubated with CBG samples as described for neutrophil elastase,
prior to steroid-binding capacity assays or SDS-PAGE. Medium from a culture of
Pseudomonas aeruginosa was used as a source of LasB (Simard et al. 2014 (link)). Indicated
amounts were added to CBG samples in 100 µL 20 mM Tris (pH 8) and
incubated (3 h at 37°C) followed by addition of 5 mM EDTA to stop
reactions, prior to steroid-binding capacity assays or SDS-PAGE.