PPIase activity was measured by a chymotrypsin-coupled assay using a synthetic peptide succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma) as previously described [51 (link)] with some modifications. Briefly, the reactions were set up in precooled 1 ml reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2) by adding 5 μl 1 mM peptide, 1 μl chymotrypsin (1 mg/ml, Sigma), and 0.5 μM purified protein. The absorbance was measured every 20 seconds for 10 min at 410 nm in a Genesys 10S UV-Vis spectrophotometer (Thermo) at 4°C.
Succinyl ala ala pro phe p nitroanilide
Manufactured by Merck Group
Sourced in United Kingdom
Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is a synthetic peptide substrate used in biochemical and cell-based assays. It serves as a specific substrate for the enzyme chymotrypsin.
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2 protocols using succinyl ala ala pro phe p nitroanilide
1
Chymotrypsin-coupled PPIase Activity Assay
2
Determination of Conidia-bound Pr1 Activity
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Conidia-bound Pr1 activity was determined according to Shah et al. (2005) as follows: 10 mg of conidia was harvested from 15-day-old culture and washed once with 1 ml of 0.03% (v/v) aq. Tween 80 and twice with 1 ml distilled water, then incubated in 1 ml of 0.1 M Tris-HCl (pH 7.95) containing 1 mM Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma-Aldrich UK) for 5 min at room temperature. After incubation, conidia were pelleted by centrifugation at 12,000 rpm for 5 min (Sanyo, Harrier 18/80 centrifuge). The supernatant (200 μl) was transferred to a 96-well flatbottom plate, optical grade (Greiner Bio-One) and absorbance measured, using a BioTek Synergy H1 multi-mode reader at 405 nm. Buffered substrate was used as a control.
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