282 Sweeney and Walker
2.2.3. Molecular Mass
The enzyme has a mol mass of approx 50,000 Da. The amino acid
composition has been published (14).
2.2.4. pH Optimum
The enzyme has a pH optimum in the range pH 7.4-7.8 in phosphate
or Tris buffer (14).
2.2.5. Assay
The assay is based on the hydrolysis of benzoyl arginine ethyl ester
(BAEE). The substrate solution is 0.05M sodium phosphate buffer, pH
7.8, 0.25 mM BAEE, and 2.5 mM dithiothreitol, at 25°C. The reaction
is followed by monitoring the change in absorbance at 253 nm with time
after addition of enzyme. A molar extinction coefficient of 1150M -1. crn -1
is used to express the results as ~tmol BAEE hydrolyzed/min at 25°C (18).
Using this assay, the enzyme is usually supplied with a specific activity
of 50-250 U/mg protein, where 1 U is defined as the amount of enzyme
that hydrolyzes 1 ~tmol of BAEE/min at pH 7.6 at 25°C.
2.2.6. Stability
Enzymic activity is rapidly lost on incubation in the presence of
oxidizing agents because of oxidation of the reactive thiol group, but
can be regenerated by treatment with dithiothreitol (14). The enzyme
is active in 6M urea (19).
2.2.7. Inhibitors
The enzyme is inhibited by any thiol reactive agent (e.g., iodoacetic
acid), by PMSF (see Section 2.1.7. for practical details), and by para-
chloromercuribenzoate (20, 21). The PMSF inhibition can be reversed
by the addition of thiol reagents, such as DTT or mercaptoethanol.
Since the reactive thiol group is easily oxidized, the enzyme is usually
used in the presence of a reducing agent, such as dithiothreitol (1-2 mM).
Some authors report that binding of calcium is an essential prerequi-
site of activity (20), and certain suppliers indicate that the enzyme
requires activation prior to use, by preincubation in 1 mM calcium
acetate, 2.5 mM DTT for 2-4 h at 25°C. The esterase activity is inhib-
ited in the presence of Co 2÷, Cu 2÷, Cd 2÷, Na ÷, and K ÷ (14). EDTA
completely inhibits activity at a concentration of 10 ~M. This is prob-
ably the result of the calcium requirement of the enzyme. When per-