Procedure 1 Pepsin, subtilisin, aminopeptidase M, prolidase

Procedure 1 pepsin, subtilisin, aminopeptidase M, prolidase This method may often be useful with proteins that can be maintained in a- largely denatured state at acid pH. The protein (usually about 10 mg per ml) may be denatured in solvents like 6 M guanidinium chloride or 8 M freshly deionised urea and dialyzed exhaustively against 5 % formic acid at room temperature at about pH 2. If cysteine or cystine residues are present, these should be reduced and alkylated ( 3.8.2) prior to enzymic hydrolysis. 

Pepsin (1 % by weight) is added to the protein in 5 % formic acid at pH 2 and hydrolysis is allowed to proceed at 40°C for at least 6 hr (overnight is often preferable). The peptide mixture is lyophilized and redissolved or suspended in 0.2 M NH4HCO3 to give a final pH between 7.5 and 8.0 (adjust with NH3 if necessary). The pepsin is denatured by the high pH. 

Subtilisin (1 % by weight) is added, and hydrolysis is allowed to proceed at 40°C for at least 6-8 hr (or overnight). The subtilisin is subsequently inhibited by incubation with 0.1 % (v/v) diisopropyl-fluorophosphate (DFP) for 1 hr at 40°C. In some instances it may be possible to omit the use of subtilisin after pepsin, or pronase or papain may be found to give better results. 

MnCl2 (0.025 M) is added to the peptide mixture obtained by the above procedure (at pH 7.5 to 8.0) to a final concentration of 0.005 M and aminopeptidase M (2000 mU per ml of peptide mixture) and prolidase (imidodipeptidase 100-200 units per ml of reaction mixture) are added hydrolysis is allowed to proceed for at least 16-24 hr. The enzymic hydrolysate is lyophilized to dryness over P2O5, redissolved in 5% acetic acid and lyophilized again. This procedure is usually sufficient to remove most of the NH3, so that analysis for the basic amino acids can be made. Repeated additions of water (or 5 % acetic acid) and lyophilization can be used to reduce the NH3 content further, if desired. 

The times of hydrolysis and the amounts of enzymes added (2 or more additions of pepsin or subtilisin may sometimes prove beneficial) 

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