Improved initial yields in C-terminal sequence analysis by thiohydantoin chemistry using purified diphenylphosphoryl isothiocyanate: NMR evidence for a reaction intermediate in the coupling reaction.
A thiohydantoin method for C-terminal sequence analysis of proteins on Zitex membranes involves derivatization of the free alpha-carboxyl group with diphenylphosphoryl isothiocyanate (DPPITC) plus treatment with pyridine to form a peptidylthiohydantoin derivative, cleavage of the thiohydantoin (TH) amino acid from the protein with potassium trimethylsilanolate, and identification of the released TH-amino acid by online reversed-phase HPLC. This automated chemistry, which was adapted to the Hewlett-Packard G 1009A sequencer, has been shown to identify two or three cycles on a wide variety of proteins, but suffers from low initial yields and instability of the DPPITC reagent. We report here an improved method for synthesis and purification of DPPITC. With this procedure the DPPITC reagent is a clear liquid that is stable at room temperature under vacuum for more than 9 months or for more than 24 months as a 1.0M solution in benzene at -20 degrees C. Using the purified reagent we were able to more than double the initial yield (from 30.7 to 72.4%) of TH-amino acid from a test protein and substantially decrease sequencer background. Examination of the reaction between DPPITC and the carboxylate of model N-terminally protected dipeptides with 31P NMR provides spectroscopic evidence for a postulated intermediate formed between the DPPITC and the peptide carboxylate. The reaction intermediate provides new insight into the coupling mechanism.
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Kenneth
K
Graham
John E
JE
Shively
D017879:Isothiocyanates / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D020539:Sequence Analysis, Protein / Q000379:methods*; D013867:Thiohydantoins / Q000737:chemistry*
D017879:Isothiocyanates; D013867:Thiohydantoins