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MODEL PEPTIDE SUBSTRATE
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Analytical work in the field of peptide and protein chemistry, especially the
elucidation of amino acid sequences involves primarily the use of proteolytic
enzymes as well as the application of selective chemical cleavage or stepwise
degradation methods to break down the amino chain of the product under
investigation. The use of such methods demands at first precise knowledge of
details of the special technique.
The Model Peptide system offered enables intensive study of such methods. The
whole kit consists of a unique, novel combination of peptides and the
appropriate free amino acids having the following structures:
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| 1. | The hexapeptide Model Substrate (MS): H-LEU-TRP-MET-ARG-PHE- ALA-OH |
2. | A series of nine model peptide standards ( Fragments of MS): a. H-LEU-TRP-OH b. H-LEU-TRP-MET-OH c. H-LEU-TRP-MET-ARG-OH d. H-LEU-TRP-MET-ARG-PHE-ALA-OH e. H-MET-ARG-PHE-ALA-OH f. H-MET-ARG-PHE-OH g. H-ARG-PHE-ALA-OH h. H-PHE-ALA-OH j. H-ARG-PHE-OH |
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The six free amino acids of which the model substrate is composed: LEU;TRP;MET;ARG;PHE;ALA |
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The Model Substrate was designed in a way that it can be cleaved by the following procedures:
ENZYMATIC DEGRADATION
1. The highly specific enzyme Trypsin, cleaves the peptide
bond between the arginine and the phenylalanine residues of MS yielding
fragments C and H.
2. Chymotrypsin cleavage occurs preferably at the bonds
between the tryptophan and methionine residues with formation of fragment A,
free alanine and fragment F, if complete hydrolysis is forced. In case of incomplete cleavage, fragments D and E can also be observed under certain conditions.
3. Aminopeptidase M splits through the entire sequence
starting at the N-terminus by subsequent removal of leucine, tryptophan and so
forth.
4. Leucineaminopeptidase also removes the amino acids
stepwise in the same manner, however with different rates.
5. Carboxypeptidase A starts hydrolysis at the C-terminus
and splits alanine and phenyalanine to form fragments D and C.
6. Carboxypeptidase B continues this degradation by removal
of arginine from fragment C to yield fragment B.
7. Pronase, which was proved to consist of a mixture of
aminopeptidases, carboxypeptidases and rather unspecific endopeptidases, cleaves
the substrate in different ways depending on the enzyme function being used.
CHEMICAL CLEAVAGE
1. Cyanogen bromide cleaves the peptide bond between the
methionine and arginine residues specifically with formation of a modified
fragment B (methionine is converted to homoserine lactone) and of fragment G.
2. N-Bromosuccinimide selectively splits the bond between
the tryptophan and methionine residues producing fragment E and a modified
fragment A (tryptophan is oxidized to dioxoindole alanine)
3. Reaction with phenylisothiocyanate at the N-terminus and
subsequent removal of leucine as phenylthiohydantoin, well known as Edman
degradation, can be repeated thus degrading the entire sequence. (see our
PTH amino acids
). In addition to each
Edman degradation step any remaining fragment (as well as the uncleaved model
substrate or fragments E, G and H) may be used to study modern methods to
determine N-terminal amino acids, such as the widely used Dansyl technique and
the Edman-Dansyl combination procedure.
The whole model peptide system may therefore be used to study, test and train
all methods describes. It might be useful for students as well as for those
research workers who start using any of these standard procedures.
In addition to the educational applicability, the model peptide system offers
a wide use for any modern research work, whenever new enzymatic or chemical
methods of peptide chain degradation are to be studied or examined. Last but
not least the system represents a very sensitive tool for advanced and
sophisticated research workers during the examination of the specificity of
newly formed proteolytic enzymes or the search for such enzymes in all kinds
of biological systems.
EXPERIMENTAL
In order to study any of the above mentioned reactions the model substrate
(MS) is cleaved and the fragments obtained are identified qualitatively or
semi-qualitatively by means of thin layer chromatography or electrophoresis in
comparison to the respective authentic model peptide standards (A to J) and
amino acids. For quantitative studies the exact amount of fragments may be
determined by ion exchange chromatography using well established equipment such
as automatic amino acid analyzers adjusted for peptide separations. In typical
examples the separation of (MS) and of all standards by means of thin layer
chromatography in two systems is demonstrated as well as the enzymatic cleavage
of (MS) by trypsin.
a) Thin Layer Chromatography:
About 50 ug of (MS) and of each peptide standard are dissolved in 0.1 ml.
methanol-water mixture (1:1;v/v). 10 ul of each solution are then pipetted on a
thin layer plate coated with silica-gel. In two solvent systems, separation is
possible as indicated in fig.1
b) Enzymatic cleavage by trypsin:
The digestion of (MS) by trypsin can be carried out at 25oC in
0.2M ammonium acetate buffer, adjusted to pH 7.0 by addition of acetic acid.
0.5 mgs. of (MS) are dissolved in 0.1 ml. MeOH and 1.0 ml. buffer solution and a
solution of 0.02 mg. of trypsin in 1 ml. buffer is added.
The incubation mixture is kept for four hours at 25oC; hereafter enzymatic degradation is stopped by addition of 0.5 ml. glacial acetic acid and the mixture is lyophilized to remove the volatile buffer. The remaining residue is dissolved in 0.5 ml. methanol/water mixture
(1:1;v/v) and 10 ul of this solution is submitted to thin layer chromatography
with the set of standards as described under (a).
or items may be ordered as a kit: 13-4879-12
MODEL PEPTIDE SUBSTRATE KIT:
13-4795-13
MODEL PEPTIDE STANDARDS (fragments only) KIT:
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