Specialists in the Isolation and Purification of Horseradish Peroxidase and a Select Range of Enzymes

Assay Procedure (Ref. No. FGAP034) - Chymotrypsin (USP / NF Units)

The method of assay is that suggested by Schwert and Takenaka in which N-Acetyl-L-Tyrosine Ethyl Ester [ATEE] is hydrolyzed at the ester linkage causing a decrease of absorbance measured at 237 nm and 25^°C.

	Chymotrypsin
ATEE + H20		N-Acetyl-L-Tyrosine + ethanol

Unit Definition

That amount of enzyme causing a decrease in absorbance at 237 nm of 0.0075 per minute at 25^°C under the specified conditions.

Reagents

1.	0.067 M Potassium Phosphate Buffer pH 7.0: Dissolve 3.53 g KH2PO4 [MM 136.09] and 7.07 g K2HPO4 [MM 174.18] in ± 800 ml distilled H2O, check pH 7.0 and dilute to 1 litre. Store buffer at 5°C.
2.	0.001 M HCl: Dilute 0.089 ml concentrated HCl [MM 36.46] to 1 litre with distilled H2O. Store on ice.
3.	Chymotrypsin Standard: Prepare a standard solution by dissolving freeze dried material in HCl (2) at a concentration of 1 mg standard / ml HCl. Immediately prior to assay dilute to yield 10 - 20 u/ml 0.001 M HCl [0.015 £ DA237/min £ 0.030].

Substrate:

Dissolve 23.5 mg N-Acetyl-L-Tyrosine Ethyl Ester (ATEE) [MM 251.3] in 100 ml buffer (1) at 70^°C. Cool rapidly and adjust A₂₃₇ to 1.2 versus buffer (1). Store at 25^°C for duration of assay.

Enzyme Sample:

Dissolve freeze dried enzyme in ice-cold HCl (2) at a concentration of 1 mg / ml. Immediately prior to assay dilute to yield 10 - 20 u/ml. [ 0.015 £ DA₂₃₇/min £ 0.030].

Procedure

Temperature = 25^°C
Wavelength = 237 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 DA237/min
Enzyme [Standard or Unknown] at zero time	0.2 ml
	3.2 ml

Record rate of decrease in absorbance at 237nm for ± 5 minutes.

Calculation

	DA 237 / min x dilution
Activity [u / mg material] =
	0.075 x 0.2 x mg enzyme / ml original solution

Where

e = molar co-efficient = 0.0075 and 0.2 is enzyme volume

Note

1. Owing to variation in substrate, the results obtained should be corrected to a USP Chymotrypsin reference standard [3].
2. Ensure that 0.015 £ DA₂₃₇/min £ 0.030.

Reference

Schwert G. W. and Takenaka Y.: (1955) Biochim, Biophys. Acta 16, 570