SOURCE. From Human Lung.
Tryptase is a serine protease purified from human lung mast cells. The enzyme is prepared by ion-exchange and affinity chromatography. Purity is approximately 95% as determined by analytical gel diffusion on Superdex 200, native PAGE and reduced SDS-PAGE. The molecular weight is approximately 140 KD comprising 4 subunits of approximately 31 to 37 KD. Tryptase has recently been shown to be an active causative enzyme in asthma (1).
Solution A; 1 mg per ml of tryptase in 0.05 M NaOAc-HAc pH 5 + 1.0 M NaCl + 0.01% NaN3.
Solution B; solution A diluted with 1 part glycerol.
At room temperature (21-23°C), solution A remained stable for 7 days. On day eight, 90% of initial specific activity remained. Solution A retained 95% of initial activity through 10 freeze thaw cycles (-20°C to room temperature) over a period of 15 days. Solution B retained 95% initial activity through 12 D temperature cycles
(-20°C to room temperature) over 25 days.
Supplied in 0.05 M NaOAc pH 5 containing 1 M NaCl + 0.01% NaN3.
Store at -20°C, stable 12 months. Protein determined by method of Bradford using BSA as standard. (4)
55-70 Units per mg of protein. One unit will hydrolyze on mM of N-benzoyl-Phe-Val-Arg-pNA
(EPC Product No. NB32) per minute at pH 8.3 at 25°C.
DETERMINATION OF HUMAN LUNG TRYPTASE ACTIVITY
Assay with N-Benzoyl-Phe-Val-Arg-pNA (EPC No. NB32) as substrate.
1.) Assay buffer; 0.1 M Tris pH 8.3 prepared at 25°C. 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 25°C
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-Pyrrolidinone. Bring to 100 ml with the tris buffer.
4.) Enzyme solution. Dissolve to 0.1 mg per ml in 0.05 M NaOAc pH 5. Maintain at 5°C.
1.) Adjust the spectrophotometer to 410 nm and the cell temperature to 25°C.
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 5ml enzyme solution and determine the increase in absorbency per minute from the
initial (1-2 min) reaction.
Calculation of Specific Activity
Vol = 3.005 ml A = 410 nm T = 25°C Light Path = 1 cm
Î 1mM pNA = 8.8 Molarity of substrate = 0.10
Units = (DA/min) (3.005)
mg 8.8 x 0.0005 mg
1.) Elrod, K., More, Wm., Am. H. Respir. Crit. Care Med. Vol., 156 pp 375-381, 1997.
2.) Schwartz, L., Lewis, R. Austin, K. , J. Biol. Chem. Vol 256 pp. 11939-11943, 1981.
3.) Smith, T., Houglands, M., Johnson, D., J. Biol. Chem. Vol 259, pp 11046-11051, 1984.
4.) Bradford, Marion, Analytical Biochemistry, 72, 248-254. (1976)
Note: All human tissue donors are screened for the following: Basic Serology Panel #1 RPR Syphilis Serology, ABO-Rh Typing HIV-1/HIV2 Antibody Screen, Hepatitis B Surface Antigen, Hepatitits B Core IgG/Igm Antibody, Hepatitis C Virus Antibody, HTLV-I Antibody. We accept only donors who are non reactive to all of the above screening.