L glutamic acid γ monohydroxamate

TGase activity was measured using a colorimetric procedure in which N-α-carbobenzoxyl-glutaminyl-glycine (N-CBZ-Gln-Gly) (Sigma, Shanghai, China) was used as the substrate [8 (link)]. Forty microliters of substrate solution (30 mmol/L N-CBZ-Gln-Gly, 100 mmol/L hydroxylamine, 10 mmol/L glutathione, 200 mmol/L Tris-HCl buffer, pH6) was added to 100 μL of TGase solution to initiate the enzymatic reaction. After 10 min, the reaction was stopped by the addition of a 40-μL terminator (1 mol/L HCl, 4 % (v/v) trichloroacetic acid, 2 % (m/v) FeCl₃ · 6H₂O), and the reaction solution was subjected to spectrophotometry analysis at 525 nm. A calibration curve was obtained using L-glutamic acid γ-monohydroxamate (Sigma, Shanghai, China). One unit of TGase was defined as that required to generate 1 μmol of L-glutamic acid γ-monohydroxamate per min at 37 °C.

Pro-TGase activation, using dispase (Worthington, NJ, USA), was performed as previously described [27 (link)]. TGase activity was measured using a colorimetric procedure in which N-α-carbobenzoxyl-glutaminyl-glycine (N-CBZ-Gln-Gly) (Sigma, Shanghai, China) was used as the substrate [6 ]. Forty microliters of substrate solution (30 mmol/L N-CBZ-Gln-Gly, 100 mmol/L hydroxylamine, 10 mmol/L glutathione, and 200 mmol/L Tris–HCl buffer at a pH of 6) was added to 100 μL of TGase solution to initiate enzymatic reaction. After 10 min, the reaction was stopped by the addition of 40 μL of terminator solution (1 mol/L HCl, 4 % (v/v) trichloroacetic acid, 2 % (m/v) FeCl₃·6H₂O) and the mixture was subjected to spectrophotometry analysis at 525 nm. A calibration curve was obtained using L-glutamic acid γ-monohydroxamate (Sigma, Shanghai, China). One unit of TGase was defined as the amount required to generate 1 μmol of L-glutamic acid γ-monohydroxamate per min at 37 °C. The results are the average of triplicate assays.