Sequential C-terminal degradation of peptides and proteins

Details Sequential C-terminal degradation of peptides and proteins Sequential C-terminal degradation of peptides and proteins

1992-05-31

1993-10-19

Housel, James C.

Chemistry: analytical and immunological testing

Peptide, protein or amino acid

Amino acid or sequencing procedure

530345, 530402, 530408, 530815, G01N 3368

Patent

active

052544753

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to the sequential degradation of peptides and proteins from the carboxy-terminus. More particularly, the invention relates to a method for the cleavage of the C-terminal thiohydantoin amino acid from the derivatized peptide which results from the use of a silylisothiocyanate as a coupling reagent in carboxy-terminal sequencing procedures. The invention also relates to derivatized and activated supports for covalent immobilization of peptides to be sequenced.


BACKGROUND OF THE INVENTION

A. Background
The development of methods for the sequential degradation of proteins and peptides from the carboxy-terminus has been the objective of several studies. See Ward, C. W., Practical Protein Chemistry - A Handbook (Darbre, A., ed.) (1986) and Rangarajan, M., Protein/Peptide Sequence Analysis: Current (1988). Such a method would complement existing N-terminal degradations based on the Edman chemistry. Edman, P., Acta. Chem. Scand. 4:283-293 (1950). The most widely studied method and probably the most attractive because of its similarity to the Edman degradation has been the conversion of amino acids into thiohydantoins. This reaction, originally observed by Johnson and Nicolet, J. Am. Chem. Soc. 33:1973-1978 (1911), was first applied to the sequential degradation of proteins from the carboxy-terminus by Schlack and Kumpf, Z. Physiol. Chem. 154:125-170 (1926). These authors reacted ammonium thiocyanate, dissolved in acetic acid and acetic anhydride, with N-benzoylated peptides to form carboxyl-terminal 1-acyl-2-thiohydantoins. Exposure to strong base was used to liberate the amino acid thiohydantoin and generate a new carboxyl-terminal amino acid. The main disadvantages of this procedure have been the severity of the conditions required for complete derivatization of the C-terminal amino acid and for the subsequent cleavage of the peptidylthiohydantoin derivative into a new shortened peptide and an amino acid thiohydantoin derivative.
Since this work was published, numerous groups have tried to reduce the severity of the conditions required, particularly in the cleavage of the peptidylthiohydantoin, in order to apply this chemistry to the sequential degradation of proteins from the carboxyl terminal end. Lesser concentrations of sodium hydroxide than originally used by Schlack and Kumpf and of barium hydroxide were found to effectively cleave peptidylthiohydantoins. See Waley, S. G., et al., J. Chem. Soc. 1951:2394-2397 (1951); Kjaer, A., et al., Acta Chem. Scand. 6:448-450 (1952); Turner, R. A., et al., Biochim. Biophys. Acta. 13:553-559 (1954). Other groups used acidic conditions based on the original procedure used by Johnson and Nicolet for the de-acetylation of amino acid thiohydantoins. See Tibbs, J., Nature 168:910 (1951); Baptist, V. H., et al., J. Am. Chem. Soc. 75:1727-1729 (1953). These authors added concentrated hydrochloric acid to the coupling solution to cause cleavage of the peptidylthiohydantoin bond. Unlike hydroxide which was shown to cause breakdown of the thiohydantoin amino acids, hydrochloric acid was shown not to destroy the amino acid thiohydantoins. See Scoffone, E., et al., Ric. Sci. 26:865-871 (1956); Fox, S. W., et al., J. Am. Chem. Soc. 77:3119-3122 (1955); Stark, G. R., Biochem. 7:1796-1807 (1968). Cromwell, L. D., et al., Biochem. 8:4735-4740 (1969) showed that the concentrated hydrochloric acid could be used to cleave the thiohydantoin amino acid at room temperature. The major drawback with this procedure was that when applied to proteins, no more than two or three cycles could be performed.
Yamashita, S., Biochem. Biophys. Acta. 229:301-309 (1971) found that cleavage of peptidylthiohydantoins could be done in a repetitive manner with a protonated cation exchange resin. Application of this procedure to 100 mmol quantities of papain and ribonuclease was reported to give 14 and 10 cycles, respectively, although no details were given. See Yamashita, S., et al., Proc. Hoshi. Pharm. 13:136-138 (1971). Stark reported that certain organic base

REFERENCES:
patent: 4582875 (1986-04-01), Ngo
patent: 4837165 (1989-06-01), Hawke
patent: 4935494 (1990-06-01), Miller
patent: 4994373 (1991-02-01), Stavrianopoulos et al.
patent: 5180807 (1991-12-01), Bailey et al.

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