Sustrate=BAPNA
I get a white solution when i mixed BAPNA(with DMSO) and buffer Tris-HCL/CaCl2
First 3 minutes the activity goes down, after 3 min stars to go up but slowly.
Is this normal?
I had only measured trypsin after 10 minutes of activity, is an end point enzyme activity, why you measured every 3 minutes? I don't understand. And I don´t remember to obtain a white solution...
BAPNA (Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride) is dissolved in DMSO (we usually use 5 mg/mL) of BAPNA). The substrate is then added (2 microL) to a 1000 microL solution (buffer+enzyme+specific effectors (please use suitable pre-incubation)). The substrate stock solution should be limpid (you have to keep it in ice and dark, and be sure it is not frozen (at 0 C DMSO will be frozen)), since BAPNA is soluble at this concentration.
Such a setup should give you the possibility to detect 3 microg of bovine trypsin easily.
You can check the reaction continuously at 410 nm.
The proposed protocol is the standard Erlanger method, still good but with some limitations:
1. BAPNA is firstly dissolved in DMSO and then brought (and kept) in the Tris-CaCl2 buffer. At this pH the auto-hydrolysis of BAPNA is not vanishable and so the substrate concentration will decrease in time. Even if the protocol uses the substrate att saturation concentration, it is not advisable to use a non-stable substrate stock solution.
2. The zymogen is dissolved in a buffer containing CaCl2. In this condition the auto-proteolysis of the zymogen (in the presence of traces of the active enzyme) is very fast (for the presence of Ca++ ions. This is the reason why "the zymogen should be prepared and used in the same day of the determination".
It is advisable to have the zymogen kept at pH=3 without any presence of Ca++. In this condition, the zymogen is very stable (you can keep it at -20C for a month).
FInally, the determination of the enzyme concentration can be obtained also kinetically, following the raise of the absorbance at 410 nm in time. Since we are using the substrate at concentrations higher than the Km, the Michaelis-Lenten velocity Vo is equal to Vmax, and since Vmax is linearly related to the concentration of Vmax, you can get the concentration of E measuring the velocity, in other words the raise of the absorbance at 410 nm in time. This method is very fast and sensitive. I use it since 1983 and still it is used nowadays in my lab. Good luck.
I am trying to validate BApNA test for trypsin for which I need a linearity graph can anyone suggest me how to make linear curve
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