Enzymatic and Chemical Deblocking of N·Yerminally Modified Proteins
315
3.2
Alcoholytic Deacetylation
N-terminally blocking of native proteins and peptides through acetylation can be
removed using incubation with trifluoroacetic acid in methanol (v/v 1:1) for 2-3
days at 47°e (Bergman el al. 1996, Gheorghe MT et al. 1997). This alcoholytic
deacetylation allows direct application of deblocked proteins and peptides to
Edman degradation with initial yields of up to 50 %.
The lower temperature gives a clear background, but also much reduced
yields. The cleavage time should be prolonged to several days or a week. The
higher temperature results in a better recovery, but the unspecific cleavage of
internal peptide bonds increases and hinders the sequence data interpretation. It
is difficult to balance between deacetylation and general alcoholysis. The deacetylation for 2 days results in sufficient cleavage and in low level of internal peptide bond cleavage. It is possible to use ethanol and isopropanol instead of methanol for de acetylation, but the initial yields are about 20-30 % lower in comparison to 60 % with methanol. For this reason short chain alcohols are optimal for
more rapid reactions. The optimal ratio TFA to methanol is 1:1. Higher concentrations of TFA causes non-specific cleavage. The yields of de acetylation are
sequence dependent, they are highest with acetyl-Ser, and very low with acetylAla. Acetyl-Ser is the most common modification in native proteins and therefore
most important for deacetylation.
3.3
Deblocking of N-Formylated Proteins
The N-formyl group of proteins can be removed, when the protein is incubated
in a dilute 0.6 M Hel at 25 °e for 24 hours. The deblocking reaction can be performed in solution or on PVDF membrane after blotting (Ikeuchi and Inoue
1988).
Milder treatment with anhydrous hydrazine vapor at -5°e for 8h may be useful to deblock the N-formylated amino acids (Miyatake et.aI1992).
3.4
Deblocking of Proteins with N-Terminal Pyroglutamic Acid
For proteins blotted onto PVDF membranes, 8h treatment with anhydrous hydrazine at 20 0 e is required to remove the N-terminal pyroglutamic acid (Miyatake
et al. 1997). During the hydrozynolysis, the pyroglutamic acid converts to Glu-yhydrazine, which can be detected using mass spectrometry or Edmandegradation. As undesired by-products, ornithin, appears after conversion of
arginine, asparagine and glutamine are detected as their hydrazide.
315
3.2
Alcoholytic Deacetylation
N-terminally blocking of native proteins and peptides through acetylation can be
removed using incubation with trifluoroacetic acid in methanol (v/v 1:1) for 2-3
days at 47°e (Bergman el al. 1996, Gheorghe MT et al. 1997). This alcoholytic
deacetylation allows direct application of deblocked proteins and peptides to
Edman degradation with initial yields of up to 50 %.
The lower temperature gives a clear background, but also much reduced
yields. The cleavage time should be prolonged to several days or a week. The
higher temperature results in a better recovery, but the unspecific cleavage of
internal peptide bonds increases and hinders the sequence data interpretation. It
is difficult to balance between deacetylation and general alcoholysis. The deacetylation for 2 days results in sufficient cleavage and in low level of internal peptide bond cleavage. It is possible to use ethanol and isopropanol instead of methanol for de acetylation, but the initial yields are about 20-30 % lower in comparison to 60 % with methanol. For this reason short chain alcohols are optimal for
more rapid reactions. The optimal ratio TFA to methanol is 1:1. Higher concentrations of TFA causes non-specific cleavage. The yields of de acetylation are
sequence dependent, they are highest with acetyl-Ser, and very low with acetylAla. Acetyl-Ser is the most common modification in native proteins and therefore
most important for deacetylation.
3.3
Deblocking of N-Formylated Proteins
The N-formyl group of proteins can be removed, when the protein is incubated
in a dilute 0.6 M Hel at 25 °e for 24 hours. The deblocking reaction can be performed in solution or on PVDF membrane after blotting (Ikeuchi and Inoue
1988).
Milder treatment with anhydrous hydrazine vapor at -5°e for 8h may be useful to deblock the N-formylated amino acids (Miyatake et.aI1992).
3.4
Deblocking of Proteins with N-Terminal Pyroglutamic Acid
For proteins blotted onto PVDF membranes, 8h treatment with anhydrous hydrazine at 20 0 e is required to remove the N-terminal pyroglutamic acid (Miyatake
et al. 1997). During the hydrozynolysis, the pyroglutamic acid converts to Glu-yhydrazine, which can be detected using mass spectrometry or Edmandegradation. As undesired by-products, ornithin, appears after conversion of
arginine, asparagine and glutamine are detected as their hydrazide.