274 Sweeney and Walker
- Inhibitors
Among the alkaline proteases, there are at least three that are inhib-
ited by diisopropyl phosphofluoridate (DFP) (18). In general, the neu-
tral proteinases are inhibited by EDTA, and the alkaline proteinases
are inhibited by DFP (4). No single enzyme inhibitor will inhibit all the
proteolytic activity in a Pronase sample.
- Inhibitors
- Experimental Procedures
3.1. DNA Isolation
When used in DNA isolation, Pronase is generally prepared as a
stock solution at about 5-20 mg/mL in water. Prior to storage at-20°C,
the solution is heated to 56°C for 15 min followed by a 1-h incubation
at 37°C. The purpose of this step is to encourage self-digestion. This
eliminates contamination with DNases and RNases. For use, the
enzyme is added to the DNA sample (in the presence of 0.5-1% SDS
to disrupt DNA-protein interactions) typically at 250-500 lag protein/
mL, 37°C, for 1-4 h.
3.2. Protein Hydrolysis
To prepare a protein hydrolysate, dissolve 0.2 grnol of protein in 0.2
mL of 0.05M ammonium bicarbonate buffer, pH 8.0 (or 0.2M sodium
phosphate pH 7.0 if ammonia interferes with the amino acid analysis).
Add Pronase to 1% (w/w), and incubate at 37°C for 24 h. To achieve
complete hydrolysis, it is usually necessary to make a further addition
of aminopeptidase M (see Chapter 18) at 4% (w/w), and incubate at
37°C for a further 18 h. The sample is then lyophilized and subjected
to amino acid analysis. When using two enzymes in this way, there is
often an increase in the background amino acids owing to hydrolysis
of each enzyme. It is therefore important to carry out a digestion blank
to correct for these background amino acids (25).
References- Hiramatsu, A. and Ouchi, T. (1963) On the proteolytic enzymes from the com-
mercial protease preparation of Streptomyces griseus (Pronase P). J. Biochem.
(Tokyo) 54(4), 462-464. - Narahashi, Y. and Yanagita, M. (1967) Studies on proteolytic enzymes
(Pronase) of Streptomyces griseus K- 1. Nature and properties of the proteolytic
enzyme system. J. Biochem. (Tokyo) 62(6), 633-641.