TR913, Tryptase

TRYPTASE

                                     NO. TR913                                  

 

SOURCE.  From Human Lung.

DESCRIPTION. 

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).

STABILITY.

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.

SPECIFICATIONS.

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.

Materials required.

     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.

Procedure

      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

References.

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.