CHAPTER 22
Enzymatic and Chemical Deblocking
of N-Terminally Modified Proteins
R. M. KAMPI and H. HIRAN02
1
Introduction
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 xray 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 Sakiyama 1992). The most common modification is the blocking with acyl- and alkylgroups. 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. The yields of these methods are
often very low and inefficient. Additionally in vitro modifications of proteins
were observed. It is important to prevent artificial blocking generated during
protein sample preparation, extraction, purification, electrophoresis or blotting
(Hirano 1997). Pure reagents, preelectrophoresis and use of thioglycolic acid as
free-radicals scavenger during extraction should be used to prevent in vitro
blocking. However, if proteins are posttranslationally blocked, the special procedure using chemical and enzymatic cleavage is required to determine the Nterminal amino acid sequence (Hirano 1997). Depending on the blocked amino
acid, several chemical or enzymatic methods can be used for removing of blockI TFH-University of Applied Sciences (Dep. of Biotechnology), Seestrasse 64, l3347 Berlin, Germany.
2 Yokohama City University, Kihara Institute for Biological Research, Maioka 641-12, Totsuka,
Yokohama, 244 Japan.
Enzymatic and Chemical Deblocking
of N-Terminally Modified Proteins
R. M. KAMPI and H. HIRAN02
1
Introduction
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 xray 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 Sakiyama 1992). The most common modification is the blocking with acyl- and alkylgroups. 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. The yields of these methods are
often very low and inefficient. Additionally in vitro modifications of proteins
were observed. It is important to prevent artificial blocking generated during
protein sample preparation, extraction, purification, electrophoresis or blotting
(Hirano 1997). Pure reagents, preelectrophoresis and use of thioglycolic acid as
free-radicals scavenger during extraction should be used to prevent in vitro
blocking. However, if proteins are posttranslationally blocked, the special procedure using chemical and enzymatic cleavage is required to determine the Nterminal amino acid sequence (Hirano 1997). Depending on the blocked amino
acid, several chemical or enzymatic methods can be used for removing of blockI TFH-University of Applied Sciences (Dep. of Biotechnology), Seestrasse 64, l3347 Berlin, Germany.
2 Yokohama City University, Kihara Institute for Biological Research, Maioka 641-12, Totsuka,
Yokohama, 244 Japan.