Bieth et al. (1974) have described an excellent assay using succinyl-(L-alanine)3-p-nitroanilide. Other suggested assays have been described: Keller and Mandl (1971), Feinstein et al. (1973), and Bieth and Meyer (1973). Schumacher and Schill (1972) describe a radial diffusion assay. The assay used in this laboratory is as follows:
Method: Derived from that of Feinstein et al. (1973) and using the more soluble substrate of Bieth et al. (1974). An increase in absorbance at 410 nm results from the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (Suc Ala3NA). One unit hydrolyzes one micromole of Suc Ala3NA per minute at 25°C and pH 8.0 under the specified conditions.
Prepare a one mg/ml solution in Tris buffer. Immediately before use dilute further to obtain a rate of 0.02-0.04 ΔA/minute.
Adjust the spectrophotometer to 410 nm and 25°C.
Pipette into each cuvette as follows:
Mix and incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration. To test cuvette add 0.2 ml of substrate, mix, and record increase A410 for 3-5 minutes. Calculate ΔA/minute from the linear portion of the curve.