HS214, Chymase

CHYMASE

NO. MK325

Lot No.   _972_______

 ____5___units

Units per mg of protein  ­­­­­ __173.6_______

mg protein per ml   ___0.125_________

 __21.7___units per ml

Volume: __0.24 ml___________

SOURCE.  Human skin.  95% pure by PAGE.

DESCRIPTION. 

Chymase is a serine protease purified from human skin mast cells.  Purity is approximately 95% by gel diffusion and SDS-PAGE.  Molecular weight »30,000.

SPECIFICATIONS.

Supplied as >0.2 mg/ml in 0.05 M NaOAc pH 5 +1 M NaCl. Store frozen at -20°C. 

150 - 170 units per mg of protein.  Protein determined by coomassie blue assay (Bradford).  One unit will hydrolyze 1.0 mmole of N-Benzoyl-L-Tyrosine-Ethyl-Ester (BTEE) per minute at pH 7.8 and 25°C.  Assay buffer is 0.30 M Tris pH 7.8 containing 1.5 M NaCl and 15% ethanol.  Use 1 to 3 mgm of enzyme.

Assay with N-Benzoyl-L-Tyrosine-Ethyl Ester as substrate.

 Materials Required:

     1.)  Buffer; 0.6 M Tris-HCl pH 8.0 containing 3.0 M NaCl at 25°C.  Dissolve 53.3 g Tris-HCl,

           31.8g Tris-base and 175.3 g NaCl with 900 ml H₂O.  Titrate to pH 8.0 at 25°C, bring to

           1000 ml with H₂O.

     2.)  Substrate;  1.0 mM.  Dissolve 31 mg N-Benzoly-L-Tyrosine-Ethyl Ester in 100 ml of

            30% (v/v) ethanol in H₂O.

     3.)  Enzyme solution.  Prepare 0.1 mg per ml in 0.05 M NaOAc pH 5.0.  Keep at 5°C  in ice

            bath.

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TECHNICAL DATA SHEET

CHYMASE

NO. MK325

Procedure

     1.)  Adjust the spectrometer to 256 nm and the cell temperature to 25°C.

     2.)  Pipette 1.5 ml of buffer and 1.5 ml of substrate into the cell.  Mix, equilibrate to 25°C in

            the cell.

     3.)  Dilute the chymase to 0.1 mg/ml.

     4.)   Add 10 ml enzyme solution; mix and record increase in A₂₅₆ nm/min for 2-4 minutes.

Calculation of Specific Activity

Vol = 3.01 ml       A = 256 nm      T= 25°C    Light Path = 1 cm        Î 1 mM  

Π1mM Tyrosine = 964 

NOTE:

                      Units  ₌    (DA₂₅₆/min) (3.01 ml)

                       mg              0.964 x 0.001 mg

REFERENCES.

1.)  Schechter, N.M, Choi, J.K., Slavin, D.A., Deresienske, D.T., Sayama, S., Dong, G.,

      Lavker, R.M., Proud, D. and Lazarus G.S., Journal of Immunology. Vol. 137, 0962-

      0970, No. 3, (1986).

2.)  Schechter, N.M., Irani, A.A., Sprows, J.L., Abernethy, J., Wintroub and Schwartz,

      L.B., Journal of Immunology, Vol 145, 2652-2661, No. 8, (1990).

Please 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, Hepatitis 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.  We have documentation on file.

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