Procedure 2 Use of insoluble enzymes

A newly described procedure for complete enzymic hydrolysis of proteins and peptides (Brown and Wold 1973) makes use of several proteases which are covalently attached to insoluble supports (Cuatrecasas et al. 1968). The advantages of using these enzymes in the insoluble form are that the enzymes may be easily removed from the products, and there is little or no hydrolysis of the enzymes themselves which reduces the corrections for controls. One disadvantage of the insoluble proteases may be that the size of the insoluble particles introduces steric factors hindering the hydrolysis of large peptides or proteins. 

Although the details of the hydrolysis procedure are yet to be published in detail (see Brown and Wold 1973), the first hydrolysis is with agarose-bound aspergillopeptidase A at pH 1.5-2.0 for 18 hr. The resulting peptide mixture is then hydrolyzed simultaneously with agarose-bound pronase and aminopeptidase M for 16 hr in borate buffer at pH 7.6, and finally with agarose-bound prolidase for 2 hr in the same buffer. It may prove helpful (not indicated in the procedure) to add the prolidase at the same time as the aminopeptidase M, since it 

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