Human Lung Tryptase Activity (83)

DETERMINATION OF HUMAN LUNG TRYPTASE ACTIVITY (83)

Assay with N-Benzoyl-Phe-Val-Arg-pNA ( EPC No. NB32) as Substrate.


Materials Required

1.     Buffer; 0.1 M Tris pH 8.3 prepared at 25ºC. Mix 0.1 M Tris-HCl into 0.1 M Tris-base until the pH =
8.3 at 25°C.

2.     Buffer; 0.05 NaOAc pH 5.0. Mix 0.05 M HAc into 0.05 M NaOAc until pH = 5.0 at 25ºC.

3.     Substrate Solution; 1.0 mM. Prepare by dissolving 68.0 mg. N-Benzoyl-Phe-Val-Arg-pNA (EPC No.
NB32) in 1 ml of 1-methyl-2-Pyrrolidone. Bring to 100 ml with the tris buffer.
 

4.     Enzyme solution. Dissolve to 1.0 mg per ml in 0.05 M NaOAc pH 5. Maintain at 5°C.
 

Procedure

1.     Adjust the spectrophotometer to 410 nm and the cell temperature to 25ºC.

2.     Pipette 2.70 ml of buffer (pH 8.3 Tris) into the cell. Add 0.3 ml substrate solution. Mix, equilibrate to
25°C in the cell.

3.     Add 5μl enzyme solution and determine the increase in absorbency per minute from the initial reaction.
 

Calculation of Specific Activity

Vol = 3.01 ml              A = 410 nm        T = 25º C       Light Path = 1 cm

Î, 1 mM Tyrosine = 0.964

Units  =  (∆A256/min) x (3.01 ml)
mg              0.964 x 0.001 mg