Proteins and
peptides can be sequenced from the carboxy terminus with
isothiocyanate reagents to produce
amino acid thiohydantoin derivatives. Previous studies in our laboratory indicated that the use of trimethylsilyl
isothiocyanate (
TMS-ITC) as a coupling
reagent significantly improved the yields and reaction conditions and reduced the number of complicating side products [Hawke et al. (1987) Anal. Biochem. 166, 298]. The present study further explores the conditions for formation of the peptidylthiohydantoins by
TMS-ITC and examines the cleavage of these peptidylthiohydantoin derivatives into a shortened
peptide and
thiohydantoin amino acid derivative.
Schizophrenia-related
peptide (Thr-
Val-Leu) was used as a model
peptide and was treated with
acetic anhydride and
TMS-ITC at 50 degrees C for 30 min, and the peptidylthiohydantoin derivatives were isolated by reverse-phase HPLC and characterized by FAB-MS. The purified derivatives were subjected to a variety of cleavage conditions, and rate constants for hydrolysis were determined. Hydrolysis with acetohydroxamate as reported originally by Stark [(1968) Biochemistry 7, 1796] was found to give excellent cleavage of the terminal
thiohydantoin amino acid, but also led to the formation of stable hydroxamate
esters of the shortened
peptide which are poorly suited for subsequent rounds of degradation. Hydrolysis with 2% aqueous
triethylamine under mild conditions (1-5 min at 50 degrees C) was found to be more suitable for carboxy-terminal sequence analysis by the
thiocyanate method. The shortened
peptide, which could be isolated and subjected to a second round of degradation, and the released
thiohydantoin amino acid are formed in good yield (90-100%). Several other small
peptides containing 15 different C-terminal
amino acid side chains were also investigated in order to examine any interfering reactions that might occur when these side chains are encountered in a stepwise degradation using the
thiocyanate chemistry. Quantitative yields of peptidylthiohydantoins were obtained for all the
amino acids examined with the following exceptions: low yields were obtained for C-terminal Glu or Thr, and no peptidylthiohydantoins were obtained for C-terminal Pro or Asp.
Asparagine was found to form cyclic
imides (64%) at the penultimate position (C-2) during hydrolysis of the peptidylthiohydantoins by 2% aqueous
triethylamine. Cleavage of C-terminal Asn under these conditions led to the formation of the expected shortened
peptide (69%), but also to the formation of a shortened
peptide (31%) with a C-terminal
amide. Problems with Glu and Thr could be solved by minimizing the reaction time with
acetic anhydride.(ABSTRACT TRUNCATED AT 400 WORDS)