294 Sweeney and Walker
absorbance at 255 nm at 25°C is measured, and the enzyme concentration
calculated using an extinction coefficient of 0.081 mM -1. cm -1.
The enzyme is supplied with a specific activity of 6000-10,000 U/mg
when this assay is used, where 1 U is defined as the amount of enzyme
that produces an increase in absorbance at 255 nm of 0.001/min at pH
7.6 and at 25°C.
2.10.6. Stability
Trypsin is stable at pH 3 at low temperatures and can be stored for
weeks without loss of activity. The lyophilized enzyme is stable for
months at 4°C. Trypsin is reversibly denatured above pH 11 (69). An
increase in temperature above pH 8 results in irreversible denatur-
ation. At pH 2-3, it is stable up to 40°C.
Trypsin is fully active in 6.5M urea, but is reversibly denatured by
8M urea (70). Urea-denatured trypsin regains activity by dialysis against
10-3M HC1 (69).
The enzyme retains activity in 30% ethanol (71), 0.1% SDS, 1M
guanidine hydrochloride, and 10% acetonitrile. Trypsin has a tendency to
autolyze at pH values >5. The addition of calcium ions (20 mM) to trypsin
solutions retards autolysis (67).
2.10. 7. Activators and Inhibitors
Trypsin is activated by calcium ions and lanthanides (72). The enzyme
is inhibited by DFP, TLCK, soybean trypsin inhibitors (73), aprotinin
(mol mass 6500 Da), leupeptin (an amino acid aldehyde, mol mass
426.6 Da), and ct-macroglobulin (74). Inhibition by benzamidine is
reversible (44).
- Experimental Procedures---Practical Details
3.1. Choice of Buffer
It is generally preferable to use a volatile buffer, particularly if the
peptides are to be separated by ion-exchange chromatography or paper
chromatography/electrophoresis, where the buffer salts may interfere,
particularly if the digested sample is concentrated or dried down at the
end of the digestion. Salt concentration is, however, not a problem if
reverse-phase HPLC is to be used to separate the peptides. The major-
ity of the enzymes described above have pH optima in the range 7.5-
8.5, and the most commonly used volatile buffers in this range are
ammonium bicarbonate (100 mM gives a pH of 8.0) or N-ethyl