Determination of t-PA in purified preparations with Chromogenic Substrate S-2288™
Tissue plasminogen activator (t-PA) is a serine proteases, which activates plasminogen by splitting a single Arg-Val bond of the plasminogen molecule. In purified systems these enzymes have been shown to hydrolyze tripeptide chromogenic substrates. The t-PA activity is thus determined by the rate at which p-nitroaniline (pNA) is released. The formation of pNA can be followed spectrophotometrically at 405 nm by using a recorder (initial rate method). The correlation between the change in absorbance per minute (ΔA/min) and the t-PA activity is linear in the 0.05 – 0.5 µkat/l or 3 – 30 U/l range. The amidolytic activity does not necessarily parallel the fibrinolytic activity for different t-PA preparations.
H-D-Ile-Pro-Arg-pNA + H2O → H-D-Ile-Pro-Arg-OH + pNA
- Chromogenic Substrate S-2288, 25 mg Art. No. S820852
Reconstitute the chromogenic substrate S-2288 (MW: 577.6) with 8.65 ml (t-PA one-chain) or 43 ml (t-PA two-chain) of distilled water.
- Tris Buffer, pH 8.4 (25°C)
Tris 12.1 g (100 mmol/l)
NaCl 6.2 g (106 mmol/l)
Distilled water 800 ml
Adjust the pH to 8.4 at 25°C by adding an appropriate amount (approximately 44 ml) of 1 mol/l HCl. Fill up to 1000 ml with distilled water. The buffer, if not contaminated, is stable for six months at 2-8°C.
- Acetic acid 20%
Acetic acid is used in the acetic-stopped method.
Purified tissue plasminogen activator is dissolved in buffer to an enzyme activity of 0.05 – 0.5 µkat/l (3 – 30 U/l). See Note. It has been advised to use a surfactant to avoid adsorption to surfaces. A final concentration of 0.1 g/l of Triton X-100 is recommended.
Initial rate method
|Incubate at 37°C||2-4 min|
|Diluted sample (20-25°C)||200 µl|
|Incubate at 37°C||2-4 min|
|Substrate (37°C)||200 µl|
Transfer sample immediately to a 1 cm semi-microcuvette (preheated to 37°C) for measurement of the absorbance change in a photometer at 405 nm and at 37°C.
The t-PA activity in the prepared tissue plasminogen activator solution is calculated from the following formulas:
µkat/l = ΔA/min x 5.21
U/l = ΔA/min x 313
In the test (600 µl) 0.25 µg (100 IU) of the porcine heart tissue plasminogen activator gives:
ΔA/min ~ 0.012 (one-chain)
ΔA/min~ 0.065 (two-chain)
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