That amount of enzyme causing the release of one micromole of p-nitroaniline per minute at 37°C and pH 8.2.
The method of assay is based on that of Szasz in Methods of Enzymatic Analysis, (Bergmeyer, H., ed., p. 715, 2nd ed. Academic Press, New York, 1974), in which the rate of increase in absorbance due to release of p-nitroaniline is measured at 405 nm and 37°C.
- 0.16 M Glycylglycine. Dissolve 20.5 mg/ml in 0.05 M Tris (free base).
- 0.016 M Magnesium chloride●6H2O. Dissolve 3.3 mg/ml in 0.05 M Tris (free base).
- 0.05 M Tris (free base).
- 120 mg Gamma-glutamyl-p-nitroanilide.
- Enzyme solution (0.1-0.2 U/ml). Dissolve enzyme in ice-cold 0.05 M Tris-HCl buffer, pH 8.2 immediately prior to assay.
- Set spectrophotometer (equipped with strip chart recorder and temperature
control) at 405 nm and 37°C.
- Into a beaker mix the following reagents. Stir at 50°C until dissolved and
adjust the pH to 8.2 with 1 M HCl or 2 M NaOH.
Glycylglycine 33 ml
Magnesium chloride 33 ml
Tris 33 ml
Gamma-glutamyl-p-nitroanilide 120 mg
- Pipette 2.90 ml of the substrate solution into a cuvette and incubate in the
spectrophotometer at 37°C for 10 minutes to attain temperature equilibration.
- Record blank rate at 405 nm.
- Add 0.10 ml of the enzyme solution to the cuvette. Mix and record the
increase in absorbance at 405 nm for 5-8 min.
- Calculate the E405 nm/min) from the linear
portion of the curve.
Activity (U/mg) = ( (E405 nm/min) (Total Vol.) (Enz. Diln.) ) / ( (9.9) (Enz. Vol.) (mg Enz. Prot/ml) )