Determination of the pH activity profile of oral gliadin-degrading enzymes.
A, Gliadin zymography. Sonicated plaque bacterial supernatant was aliquoted into eight equal fractions. Each fraction containing 90 µg protein was loaded onto a gliadin zymogram gel. After electrophoresis and renaturing the gel was cut and individual lanes were developed in 20 mM Tris buffers exhibiting pH values from 3 to 10. Far left lane: molecular weight standard. B, Z-YPQ-hydrolysis. Plaque bacteria were suspended 20 mM Tris buffers varying in pH from 2 to 10 to a final OD620 of 1.2. Suspensions were mixed with Z-YPQ-pNA (200 µM). Substrate hydrolysis was monitored spectrophotometrically at 405 nm. A representative graph of 3 independent experiments is shown.