Preparation of Chemically Defined ɛN‐Acetylated Trypsin

Acetylation of trypsin by acetic anhydride has been reinvestigated: it leads to a heterogeneous preparation, which can be fractionated by ammonium sulphate precipitation followed by chromatography on DEAE‐cellulose. The degree of substitution of ɛ and α‐amino groups, and that of phenolic hydroxyl groups was examined in fractions at different stages of purification, and it has been found that while all the ɛ‐aminogroups of lysine are acetylated the α‐aminogroup of the N‐terminal isoleucine remains free. The exposed tyrosyl residues of trypsin are also substituted, but the resulting ester bond is not very stable: these residues are slowly deacetylated on aging, even at slightly alkaline pH. This deacetylation brings about a decrease in the esterolytic activity of the enzyme. The final, homogeneous N ‐ɛ‐acetyltrypsin obtained exhibits the same activity as native trypsin towards synthetic substrates.