Assay Procedure (Ref. No. FGAP037) - Trypsin
(USP / NF Units)
The method of assay is that suggested by Schwert and Takenaka in
which N-benzoyl-L-arginine ethyl ester [BAEE] is hydrolysed at the ester linkage causing
an increase of absorbance measured at 253nm and 25°C.
|
Trypsin |
|
| BAEE + H20 |
 |
N-benzoyl-L-arginine + ethanol |
Unit Definition
That amount of enzyme causing an increase in absorbance at 253 nm
of 0.003 per minute at 25°C under the specified conditions.
Reagents
| 1. |
0.001M HCl:
Dilute 0.089ml concentrated HCl [MM 36.46] to 1 litre with
distilled H2O. Store on ice.
|
| 2. |
0.067 M Potassium Phosphate Buffer pH7.6:
Dissolve 1.17g KH2PO4 [MM136.09] and 10.08g
K2HPO4 [MM174.18] in distilled H2O, check pH7.6 and
dilute to 1 litre.
|
| 3. |
Trypsin Standard:
Prepare standard solution by dissolving freeze dried material in
HCl [1] at a concentration of 1 mg Standard / ml HCl. Immediately prior to assay dilute to
yield ± 40 u / ml ice-cold HCl [1] [DA253 / min ± 0.024]. |
Substrate:
Dissolve 8.6 mg BAEE . HCl [MM342.82] / 100 ml buffer [2] and
adjust A253 to 0.575 versus buffer [2]. Store at room temperature.
Enzyme Sample:
Dissolve freeze dried enzyme in ice-cold HCl [1] at a
concentration of 5 mg / ml. Immediately prior to assay dilute to yield ± 40 u/ml ice-cold
HCl [1]. [DA253
/ min ± 0.024].
Procedure
Temperature = 25°C
Wavelength = 253 nm
Light path = 1 cm
Cuvette volume = 3.2 ml
Sample volume = 0.2 ml
Into a 10 mm quartz cuvette pipette the following:
| Substrate |
3.0 ml |
| Equilibrate at 25°C and monitor DA253 / min. |
|
| Enzyme [Standard or Unknown] at zero time |
0.2 ml |
|
3.2 ml |
Record rate of increase in absorbance at 253 nm for ± 5 minutes
Calculation
|
DA253
/ min x dilution |
| Activity [u/mg material] = |
 |
|
0.003 x 0.2 x mg enzyme/ml original solution |
[Where e , the molar extinction coefficient = 0.003 and sample
volume = 0.2 ml]
Note
- Due to variations in the quality of the substrate, it is necessary
to calibrate the assay system using a USP Trypsin reference standard.
- Ensure that DA253 / min ± 0.024.
Reference
Schwert G.W. and Takenaka Y. : (1955) Biochim. Biophys Acta 16,
570 |
Specialists in the Isolation and Purification of Horseradish
Peroxidase and a Select Range of Enzymes