Deacetylation and Internal Cleavage of Polypeptides for N-Terminal Sequence Analysis

Abstract

Proteins with a blocked N-terminus are common. Frequently the modification involves an acetyl-, formyl- or pyroglutamyl-moiety coupled to the α-amino group and direct sequence analysis by Edman degradation is not possible. Several enzymatic and chemical methods to remove the blocking group have been suggested (cf. Tsunasawa and Hirano, 1993), but they often suffer from poor yields and a large extent of undesirable peptide bond cleavage. Acetylation represents the most frequent N-terminal modification and is found in alcohol dehydrogenases among many other proteins. To circumvent the conventional approach to sequence analysis of blocked proteins (i.e. proteolytic cleavage, HPLC of fragments and internal sequence analysis) we have tested direct chemical deacetylation using a mixture of trifluoroacetic acid and methanol (Gheorghe et al., 1995). In this manner, drawbacks as high protein consumption, long handling times and inaccessibility of the N-terminal fragment to Edman degradation, are avoided. The protocol has been applied to both a synthetic peptide corresponding to the N-terminal segment of horse liver alcohol dehydrogenase and to the intact protein.