Enzymatic and Chemical Deblocking of N-Terminally Modified Proteins
305
To determine the efficiency of deblocking and optimize the cleavage conditions,
fluorescence labelled amino acids such as Ac-Met-MCA {MCA-{4-methylcoumaryl-7-amide}, Met-MCA, Pyr-MCA and peptides such as Ac-TVG, AcGVDEK, Ac-DEVD-MCA were used_ It is very important to find the optimal balance between cleavage time and the sequence initial yields. If longer time and
higher temperature (50-80°C) were used, the deacetylation was more efficient,
but high background level significantly decreased the sensitivity and made identification of amino acid impossible.
Since acetylation is one of the most common modifications in proteins, a variety of samples with N-terminally acylated amino acids were investigated, such as
cytochrome c, lentinus protease inhibitor, ovalbumin, superoxide dismutase,
neurotensin and a-MSH. The following acylated amino acids occur in these proteins and peptides: Ac-Ala, Ac-Gly, Ac-Ser Pyr-Glu and Ac-Met. Additionally,
formyl- and myristoyl groups can be removed using the above procedure (personal communication Dr. S. Tsunasawa). We carried through similar tests with
nonblocked proteins to determine the yields of deblocking reaction. Depending
on blocking group and length of the polypeptide chain, the time of cleavage differed from hours to several days. The deblocking aminopeptidase removed acylated amino acids very slowly, and after cleavage of the first blocked amino acid,
the speed of protein degradation rapidly increased. At present, it is impossible to
remove only the N-terminal blocking group with DAP. DAP can cleave all peptide
bonds sequentially from N-terminus, except the peptide bond at the N-terminal
side of X-Pro sequence. The optimized conditions for digestion with DAP are:
50mM ethylmorpholine buffer pH 8.0 containing 0.1 % CoCI2, temperature
50°C. The time of the cleavage varies from hours for small proteins to 2 days for
large proteins. The optimized enzyme:substrate molar ratio increased from 1:100
for small peptides to 1:1 for large proteins. The treatment with DAP under optimized conditions results in sufficient deblocking with initial yields up to 50 %.
However, the initial yields in the sequencer for some large proteins were below
5 %. Fig. 1 shows the amino acid sequence analysis after deblocking of 100 pmol
of neurotensin. The conditions were: 50mM N-ethylmorpholine pH 8.0, time 24h,
temperature 50°C and substrate: enzyme ratio 1:1. Because the amount of the
used enzyme is relatively high, not only the sequence of neurotensin after cleavage one amino acid before proline (Lys-Pro-Arg-Arg-Pro-Tyr .. ), but also the
sequence of DAP was analyzed in the run (Met-Tyr-Asp-Tyr-Glu-Leu ... ). The first
four amino acids PyrLeu-Tyr-Glu-Asn cannot be analysed, because the cleavage
with DAP is very fast and only appearance of proline in the sequence can stop
this rapid digestion. More advantageous is to apply blocked, acetylated-DAP,
whose sequence does not appear in the chromatogram, but unfortunately such
enzyme is commercially not available.
Although the cleavage can be performed in the solution or after elution of proteins from the PVDF membrane, unfortunately the direct cleavage on PVDF
membrane was not successful.
We found that proteins are efficiently eluted from the PVDF membranes with
70 % acetic acid and sonication for 1/2 hour (Table 22.2, Fig. 4). The extraction
efficiency is higher than that of 0.1 M ammonium bicarbonate in 40 % acetonitrile, 0.1 M ammonium bicarbonate in 40 % acetonitrile and 2 % /3-mercapto-
305
To determine the efficiency of deblocking and optimize the cleavage conditions,
fluorescence labelled amino acids such as Ac-Met-MCA {MCA-{4-methylcoumaryl-7-amide}, Met-MCA, Pyr-MCA and peptides such as Ac-TVG, AcGVDEK, Ac-DEVD-MCA were used_ It is very important to find the optimal balance between cleavage time and the sequence initial yields. If longer time and
higher temperature (50-80°C) were used, the deacetylation was more efficient,
but high background level significantly decreased the sensitivity and made identification of amino acid impossible.
Since acetylation is one of the most common modifications in proteins, a variety of samples with N-terminally acylated amino acids were investigated, such as
cytochrome c, lentinus protease inhibitor, ovalbumin, superoxide dismutase,
neurotensin and a-MSH. The following acylated amino acids occur in these proteins and peptides: Ac-Ala, Ac-Gly, Ac-Ser Pyr-Glu and Ac-Met. Additionally,
formyl- and myristoyl groups can be removed using the above procedure (personal communication Dr. S. Tsunasawa). We carried through similar tests with
nonblocked proteins to determine the yields of deblocking reaction. Depending
on blocking group and length of the polypeptide chain, the time of cleavage differed from hours to several days. The deblocking aminopeptidase removed acylated amino acids very slowly, and after cleavage of the first blocked amino acid,
the speed of protein degradation rapidly increased. At present, it is impossible to
remove only the N-terminal blocking group with DAP. DAP can cleave all peptide
bonds sequentially from N-terminus, except the peptide bond at the N-terminal
side of X-Pro sequence. The optimized conditions for digestion with DAP are:
50mM ethylmorpholine buffer pH 8.0 containing 0.1 % CoCI2, temperature
50°C. The time of the cleavage varies from hours for small proteins to 2 days for
large proteins. The optimized enzyme:substrate molar ratio increased from 1:100
for small peptides to 1:1 for large proteins. The treatment with DAP under optimized conditions results in sufficient deblocking with initial yields up to 50 %.
However, the initial yields in the sequencer for some large proteins were below
5 %. Fig. 1 shows the amino acid sequence analysis after deblocking of 100 pmol
of neurotensin. The conditions were: 50mM N-ethylmorpholine pH 8.0, time 24h,
temperature 50°C and substrate: enzyme ratio 1:1. Because the amount of the
used enzyme is relatively high, not only the sequence of neurotensin after cleavage one amino acid before proline (Lys-Pro-Arg-Arg-Pro-Tyr .. ), but also the
sequence of DAP was analyzed in the run (Met-Tyr-Asp-Tyr-Glu-Leu ... ). The first
four amino acids PyrLeu-Tyr-Glu-Asn cannot be analysed, because the cleavage
with DAP is very fast and only appearance of proline in the sequence can stop
this rapid digestion. More advantageous is to apply blocked, acetylated-DAP,
whose sequence does not appear in the chromatogram, but unfortunately such
enzyme is commercially not available.
Although the cleavage can be performed in the solution or after elution of proteins from the PVDF membrane, unfortunately the direct cleavage on PVDF
membrane was not successful.
We found that proteins are efficiently eluted from the PVDF membranes with
70 % acetic acid and sonication for 1/2 hour (Table 22.2, Fig. 4). The extraction
efficiency is higher than that of 0.1 M ammonium bicarbonate in 40 % acetonitrile, 0.1 M ammonium bicarbonate in 40 % acetonitrile and 2 % /3-mercapto-