Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae
Fig 5
Enzyme assay for characterization of Rhi o 1 as aspartic protease.
(A) In-gel: 0.02% Gelatin zymography of purified nRhi o 1 (lane 1) and rRhi o 1 (lane 2) in 7% polyacrylamide gel. Due to protease activity colorless zones appeared at region corresponding to Rhi o 1 position on gel and remaining gel stained blue with CBB-R250 due to presence of gelatin. (B) In-sol: Spectrophotometric assay of aspartic protease with BSA as substrate. 100 nM Rhi o 1 catalyzed degradation of 2% BSA in presence of 50 mM sodium citrate (pH 3.2). After terminating reaction with perchloric acid, precipitated proteins were removed and absorbance of the clear supernatant containing degraded products of BSA was taken at 280 nm. Purified nRhi o 1 (blue line) and rRhi o 1 (red line) displayed time dependent increase in A280 of the supernatant.