MODEL PEPTIDE SUBSTRATE

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:

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.

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.

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 25^oC 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 25^oC; 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).

Product Number	Product Name	Sold in quantities of:
12-4350-02	H-LEU-TRP-MET-ARG-PHE-ALA (MS)	50 mg
12-4351-02	H-LEU-TRP (fragment A)	50 mg
12-4352-02	H-LEU-TRP-MET (fragment B)	50 mg
12-4353-02	H-LEU-TRP-MET-ARG (fragment C)	50 mg
12-4354-02	H-LEU-MET-MET-ARG-PHE (fragment D)	50 mg
13-4794-02	H-MET-ARG-PHE-ALA (fragment E)	50 mg
13-4811-02	H-MET-ARG-PHE (fragment F)	50 mg
01-0268-02	H-ARG-PHE-ALA (fragment G)	50 mg
16-5916-02	H-PHE-ALA (fragment H)	50 mg
01-0315-02	H-ARG-PHE (fragment J)	50 mg

or items may be ordered as a kit:

13-4879-12    MODEL PEPTIDE SUBSTRATE KIT:
50 mgs. of MS plus 10 mgs. of each of the standards and 1 gm. each of Leu;Trp;Met;Arg;Phe;Ala

13-4795-13    MODEL PEPTIDE STANDARDS (fragments only) KIT:
50 mgs. of each of the nine standards