Pyridylethylation of cysteine residues in proteins.
A procedure is described for using an inert polyvinylidene difluoride type membrane (e.g., ProBlott) as a matrix for on-membrane gas-phase pyridylethylation and as a desalting (cleanup) support following the solution phase pyridylethylation of cysteine residues in proteins. As a desalting support for 200 pmol of cardiotoxin, the membrane-derived sample gave a better recovery of phenylthiohydantoin (PTH)-amino acids during N-terminal sequencing compared to the sample obtained by rpHPLC. Using on-membrane gas-phase pyridylethylation, followed by replacement of glass-fiber filter disc and membrane washing/drying in vacuo, it was possible to eliminate the broad background peak that coeluted with PTH-Ala in the HPLC chromatogram completely. Using this modified procedure, there was no evidence of N-terminal alkylation of the amino-termini of proteins.
Kao
M C
MC
Bioprocessing Technology Unit, National University of Singapore.
Chung
M C
MC
D000478:Alkylation; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry*; D005465:Fluorocarbon Polymers
D003545:Cysteine; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D011725:Pyridines; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers