Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Patent
1993-07-26
1995-07-11
Redding, David A.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
530402, 530427, G01N 3368, C07K 100
Patent
active
054320925
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method, which may be automated, for the C-terminal sequencing of peptides in which a novel combination of reagents is utilized. More particularly, the invention relates to C-terminal peptide sequencing with a reagent comprising diphenyl phosphoroisothiocyanatidate and pyridine.
BACKGROUND OF THE INVENTION
A. The Prior Art
The large number of cloned gene products and the widespread use of DNA sequencing to determine the primary structure of peptides, has increased the need for carboxy-terminal (C-terminal) sequencing. C-terminal sequencing complements existing N-terminal degradations based on Edman chemistry (Edman, P. Acta. Chem. Scand. 4:283-293 (1950)). Although many methods have been proposed (see Rangarajan, M., Chemical methods of amino acid sequence analysis from carboxy terminal end. In Protein/Peptide sequence Analysis: Current Methodologies (Brown, A. S., Ed.) pp. 135-144, CRC Press, Boca Raton, Fla. (1988) and Ward, C. W., Carboxyl terminal sequence analysis. In Practical Protein Chemistry--A Handbook (Darbre, A., Ed.) pp. 491-525, John Wiley and Sons, Ltd. (1986)) the thiocyanate method, first described by Schlack and Kumpf, Z. Physiol. Chem. 154:125-170 (1926), has been the most widely studied and the most attractive because of its similarity to the Edman degradation. This method involves reaction of a protein or peptide with isothiocyanate reagents, in the presence of acetic anhydride, to form a C-terminal thiohydantoin amino acid. The derivatized amino acid is then hydrolyzed to yield a shortened polypeptide and a thiohydantoin amino acid. As the thiohydantoin amino acids have similar UV absorption spectra and equivalent extinction coefficients as the phenylthiohydantoin amino acids formed during the Edman degradation, the sensitivity of the thiocyanate method is expected to be similar to that of current N-terminal methods (10-200 pmol, 20-30 cycles). Historically, 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 hydrolysis of the derivatized amino acid to yield a shortened peptide and thiohydantoin derivative. Although several groups have tried to reduce the severity of the hydrolysis conditions (Waley, et al. J. Chem. Soc. 1951:2394-2397 (1951); Kjaer, et al. Acta Chem. Scand. 6:448-450 (1952); Turner, et at. Biochim. Biophys. Acta. 13:553-559 (1954), it was not until the introduction of acetohydroxamate (Stark, G. R. Biochemistry 7:1796-1807 (1968) as a mild and rapid cleavage reagent, that the hydrolysis problem appeared solved. The introduction of trimethylsilylisothiocyanate (TMS-ITC) (see U.S. Pat. No. 4,837,165) improved the yield of thiohydantoin formation and reduced the number of complicating side products. The repetitive yields were low, limiting the number of degradation cycles to 2 or 3 residues. Certain amino acids were reported to be unable to form thiohydantoins (Hawke, et al. Anal. Biochem. 166:298-307 (1987); Miller, et al., Techniques in Protein Chemistry (Hugli, T. E., Ed.) pp. 67-78, Academic Press, Inc. (1989)).
Recent work, Bailey, et al. Biochemistry 29:3145-3156 (1990), found that hydrolysis with acetohydroxamate led to the formation of a shortened peptide with a stable hydroxamate ester at the C-terminus, thereby preventing further degradation and explaining the low repetitive yields obtained with this reagent (Miller, supra; Meuth, et al., Biochem. 21:3750-3757 (1982)). Hydrolysis with dilute aqueous triethylamine was found to lead to the quantitative formation of a thiohydantoin amino acid and a shortened peptide capable of continued degradation. Additional work (Bailey, supra) addressed the generality of the thiocyanate method by examining the reaction of TMS-ITC with model peptides containing most of the naturally occurring amino acids. Problems were identified when Pro, Asp, Glu, Thr, and Asn were encountered during the degradation. Minimization of the reaction time with acetic anhydride was found to a
REFERENCES:
patent: 4837165 (1989-06-01), Hawke
patent: 5041388 (1991-08-01), Boyd et al.
patent: 5049507 (1991-09-01), Hawk et al.
patent: 5180807 (1993-01-01), Bailey et al.
Kenner, G. W. et al., J. Chem. Soc. 673-678 1953.
Bailey Jerome M.
Shively John E.
City of Hope
Irons Edward S.
Redding David A.
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