The Research Of C-terminal Sequencing Analysis And Biological Activity Of HWAP-Ⅰ

Sequence analysis of proteins and peptides is an important technique in protein chemistry. The chemical degradation method established by Edman in 1950 has been a routine approach for N-terminal sequencing and is widely employed by automated protein sequencer. The C-terminal sequence is as important as N-terminal sequence in protein structure, and sequencing from C-terminus appears more and more important. Not only sequence information from the c-terminus of proteins and peptides is of especial interest in the investigation of N-terminally blocked proteins and peptides, but also C-terminal sequence analysis can facilitate the production of more specific probes for gene cloning. Among numerous strategies for C-terminal protein sequencing (iso) thiocyanate method established by Schlack-Kumpf in 1926 appears to be a promising approach because of the Edman degradation in mechanism. However, due to the poor reactive earboxyl group of C-terminus, it is difficult to derivatize peptides to peptidyl thiohydantoins for C-terminus, and the Schlack-Kumpf degradation based on thiohydantoin procedure has not yet been developed to such an efficient method as Edman degradation. But in recent developments, especially in derivatizing reagents, have demonstrated that this method appears very promising in the field of protein C-terminal sequence analysis in future. Furthermore, with the significant improvements of mass spectrometry technologies, which facilitate analysis of largebiological molecules. Application of mass spectrometry technique for C-terminal sequence determination is a kind of new strategy, and now many achievements have been made in recent years. Mass spectrometry technique will play more and more role in the field of sequence analysis.Standard amino acid thiohydantoins are required as reference standard for development of C-terminal protein sequencing based on the ghiohydantoin procedure. All of 19 amino acid thiohydantoins were synthesized by using free amino acids as starting materials, acetic anhydride as the carboxyl group activating reagent and TMS-ITC as the derivatizing reagent: The proline thiohydantoin was prepared by using ammonium thiocyanate (NH4SCN) as the derivatizing reagent. Products were purified by reversed phase HPLC and characterized by ultraviolet spectra.We spend much rime on the derivatizing reagent which isextremely crucial to this method. Schlack-Kumpf degradationgenerally involves: (1) Carboxyl activation: the carboxylgroup is activated by acetic anhydride and then' producepeptidyl oxazolinone; (2) Coupling reaction : the reaction of peptidyl oxazolinone with one of the above derivztzing reagents produces peptidyl isothiocyanate; (2) Cyclization reaction: Cyclization reaction automatically forms peptidyl thiohydantoin; (4) Cleavage reaction:This method is thought to be a promising approach tochemical C-terminal seqencing of peptides and proteins. The derivatizing reagent is most crucial to this method. Two derivating reagents, triphenylgermanium isothiocyanate and tribenzylsilyl isothiocyanate, has been synthesized and applied to C-terminal peptide sequencing, four C-terminal residues of a model peptid at low nanomole levels.The initial yield is 60. 8% and the repetitive yield is 76. 7% using TBS-ITC, The initial yield is 68.8% and the repetitive yield is 81.4% using TPGe-ITC.