Enzymatic and Chemical Deblocking of N-Terminally Modified Proteins

Abstract

Proteins are complicated structures, forming three dimensional folds of the linear polypeptide chain, which is specific and unique for each of the various proteins. The specifity is fully imprinted in the amino acid sequence and firstly the primary structure has to be established. Additionally, the knowledge of amino acid sequence is the basis for identification of purified proteins, for construction of oligonucleotides and further isolation of the corresponded genes, for preparation of antibodies and for correct interpretation of structural analysis data as X-ray data and folding of the linear structure. Sequence information can be obtained by direct amino sequence analysis using pmol amounts of protein. Unfortunately 80% of all intracellular soluble proteins from eukaryotic cells and a few proteins in prokaryotes are N-terminally blocked (Kraft 1997) and cannot be sequeced by direct Edman degradation (Aitken 1990, Tsunasawa and Saki-yama 1992). The most common modification is the blocking with acyl- and alkyl-groups. Tsunasawa and Hirano (1993) described commonly blocked amino acids in polypeptide chain (see Table 22.1). The sequence data can be obtained from the N-terminally blocked proteins only after removing these blocking groups. Thus, there is a need for a simple and rapid technique for obtaining sequence information of blocked proteins. Most techniques proposed for N-terminally blocked polypeptides involve internal cleavage of the respective proteins and subsequent sequencing of the single peptide chains.