280 Sweeney and Walkeralso been reported C terminal to Met, Ileu, Ser, Thr, Val, His, Gly, and
Ala residues. The nature of neighboring residues can affect the rate of
cleavage of a particular bond. Irrespective of the nature of X, X--Pro
bonds are not cleaved (6).
2.1.3. Molecular Mass
~-Chymotrypsin (chymotrypsin A) has a mol mass of about 25,000
Da and contains 241 amino acid residues. The molecule has three
polypeptide chains (A chain--13 residues; B chain--131 residues;
and C chain--97 residues) linked by disulfide bridges. The amino acid
sequence is known (7).
2.1.4. pH Optimum
The enzyme has a pH optimum between 7.5 and 8.5 (2).
2.1.5. Assay
The assay is based on measuring the esterolytic activity of the enzyme.
The decrease in absorbance caused by the hydrolysis of the ester link-
age of N-acetyl-L-tyrosine ethyl ester (ATEE) is measured spectropho-
tometrically at 237 nm.
ATEE (2.8 mL of 2 mM in 0.1M phosphate buffer, pH 7.0) is incubated
at 25°C. At time zero, 20 laL of enzyme (diluted in 1 mM HC1) are added.
The change in absorbance is monitored with time at 237 nm. The enzyme
is usually supplied with a specific activity of 9000-11,000 ATEE U/
mg when this assay is used. One ATEE unit is defined as the amount
of enzyme that causes a decrease in absorbance of 0.001/min (8,9).
Alternatively, an assay based on hydrolysis of Suc-(Ala)2-Pro-Phe-
4-nitroanilide can be used. The reaction is monitored at 4 l0 nm. Using
this assay, the enzyme is usually supplied with a specific activity of
60-70 U/mg, where 1 U is defined as the amount of enzyme that
hydrolyzes 1 ~tmol of substrate/min at pH 7.0, and 25°C.
2.1.6. Stability
The enzyme functions in the presence of 2M guanidine hydrochlo-
ride (10) and 0.1% SDS. Stock solutions ( 10 mg/mL) in 1 mM HC1 can
be stored frozen or for a number of days at 4°C. When diluted for use
to 1 mg/mL in 1 mM HC1 (since autolysis occurs at high pH), the
presence of 2 mM Ca 2÷ helps stabilize (or possibly activate) the enzyme.
The lyophilized enzyme is stable for years at 4°C.