Chymotrypsin Assay

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Chymotrypsin Assay

Method

The reaction velocity is determined according to Hummel (1959) by measuring an increase in absorbance at 256 nm resulting from the hydrolysis of benzoyl-L-tyrosine ethyl ester. One unit hydrolyzes one micromole of benzoyl-L-tyrosine ethyl ester (BTEE) per minute at pH 7.8 and 25°C under the specified conditions.

Reagents

0.08 M Tris⋅HCl buffer, pH 7.8 containing 0.1 M calcium chloride
0.00107 M Benzoyl-L-tyrosine ethyl ester (BTEE) in 50% w/w methanol (63 ml absolute methanol added to 50 ml reagent grade water)
0.001 N HCl

Enzyme

Dissolve enzyme at one mg/ml in 0.001 N HCl. Dilute in 0.001 N HCl to 10-30 μg/ml for assay.

Procedure

Adjust the spectrophotometer to 256 nm and 25°C.

Pipette into cuvettes as follows:

0.08 M Tris⋅HCl buffer, pH 7.8 with 0.1 M CaCl₂ 1.5 ml

0.00107 M BTEE 1.4 ml

Incubate in spectrophotometer at 25°C for 4-5 minutes to achieve temperature equilibrium and record blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record increase in absorbance at 256 nm for 4-5 minutes. Calculate ΔA₂₅₆/min from the initial linear portion of the curve.

Calculation

where 964 is the extinction coefficient of BTEE at 256 nm

\frac{\text{Units}}{\text{mg}}\text{ = }\frac{\frac{\Delta \text{A}_{256}}{\text{min}}\text{ x 1000}}{\text{964 x }\frac{\text{mg}}{\text{ml}} \text{ in the reaction mixture}}

Chymotrypsin Products

Description

Chymotrypsin, Alpha, TLCK Treated, Sequencing Grade

Source:

Bovine Pancreas

Three time crystallized and treated with 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK) to inhibit trypsin activity [Shaw, et al., Biochemistry, 4, 2219 (1965)]. Dialyzed to remove autolysis products and low molecular weight contaminants. Supplied as lyophilized powder.

Store at 2-8°C.

…

Activity

≥40 u/mgP

Certificate of Analysis

Code

CDSEQ

Product details

LS02130

4x25 ug

$180.00

LS02132

4x100 ug

$472.00

Description

Chymotrypsin, Alpha, TLCK Treated

Source:

Bovine Pancreas

Three times crystallized and treated with 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK) to inhibit trypsin activity [Shaw, et al., Biochemistry, 4, 2219 (1965)]. Dialyzed against 1 mM HCl to remove autolysis products and low molecular weight contaminants. Supplied as a dialyzed, lyophilized powder.

Store at 2-8°C.

…

Activity

≥45 units per mg protein

Certificate of Analysis

Code

CDTLCK

Product details

LS001430

25 mg

$31.00

LS001432

100 mg

$75.00

LS001434

1 gm

$584.00

LS001438

Bulk

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Description

Chymotrypsin, Alpha, Purified

Source:

Bovine Pancreas

Chromatographically prepared by the procedure of Yapel, et al., J. Amer. Chem. Soc., 88, 2573 (1966). A lyophilized powder.

Store at 2-8°C.

…

Activity

≥45 units per mg protein

Certificate of Analysis

Code

CDS

Product details

LS001475

100 mg

$76.00

LS001479

1 gm

$578.00

LS001477

Bulk

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Description

Chymotrypsin, Alpha

Source:

Bovine Pancreas

Three times crystallized alpha chymotrypsin which is an activation product of a 3X crystallized zymogen. Dialyzed against 1mM HCl and lyophilized.

Store at 2-8°C.

…

Activity

≥45 units per mg protein

Certificate of Analysis

Code

CDI

Product details

LS001448

250 mg

$44.00

LS001450

1 gm

$127.00

LS001451

10 gm

$955.00

LS001453

Bulk

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Description

Chymotrypsin, Alpha

Source:

Bovine Pancreas

Crystallized as zymogen and activated. Dialyzed against 1mM HCl and lyophilized.

Store at 2-8°C.

…

Activity

≥35 units per mg protein

Certificate of Analysis

Code

CDAG

Product details

LS001333

1 gm

$38.00

LS001334

10 gm

$225.00

LS001332

Bulk

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0.08 M Tris⋅HCl buffer, pH 7.8 with 0.1 M CaCl₂	1.5 ml
0.00107 M BTEE	1.4 ml

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Chymotrypsin - Assay

Chymotrypsin Assay

Chymotrypsin Products