314
OH
I
R yH2 R f R
CH3-C-NH-CH-C-NH-CH-CCH2 0
R
0
+
II
II
I
II
H3N--CH-C-NH-CH-C! PlTe
Sequencing
R. M. KAMP and H. HIRANO
N-acetylated Protein
Fig. 22.6. Mechanism of deblocking reaction in N-acetylated proteins by trifluoroacetic acid
(Wellner et al. 1990)
vapor, the time of cleavage is decisive for sequencing yields. The yields of PTH
increase, when the reaction is longer than 2h, but too long reaction causes higher
formation of reaction by-products which hinder identification of PTH amino
acids. Reaction by-products may be generated primarily through unspecific
cleavage of polypeptide chain (Hulmes and Pan 1991).
Although the mechanism of the deblocking reaction is not known, Wellner
suggested that N-O acyl shift may be involved (see Fig. 22.6).
Takakura et. al. 1992 described other treatment with TFA for deblocking of
acetylated proteins. Proteins separated by 2D-electrophoresis and blotted onto
the PVDF membrane were exposed to vapour TFA for 16h at 65°C. After deblocking reaction the PVDF membrane can be used directly for automated
sequence analysis (Takakura et al. 1992).