On-line post-column fluorescence detection for N-terminal tyrosine-containing peptides in high-performance liquid chromatography.
A detection system based on on-line post-column fluorescence derivatization is described for the determination of N-terminal tyrosine-containing peptides by reversed-phase high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives by reaction with hydroxylamine, cobalt (II) and borate after peptide separation on a reversed-phase column (TSKgel ODS-120T) followed by passage through an ultraviolet absorbance detector. The reaction system permits the fluorescence detection at 435 nm (emission) with excitation at 335 nm for N-terminal tyrosine-containing synthetic peptides in as little as picomole amounts. The facile fluorescence detection of N-terminal tyrosine-containing fragments produced from methionine-enkephalin by enzymatic degradation using a rat brain homogenate was achieved by comparison with the ultraviolet absorption detection at 215 nm.
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
M
M
Ohno
M
M
Kai
Y
Y
Ohkura
D000818:Animals; D001923:Brain Chemistry; D002540:Cerebral Cortex / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004744:Enkephalin, Methionine / Q000032:analysis; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008297:Male; D010455:Peptides / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000032:analysis*
D007202:Indicators and Reagents; D010455:Peptides; D014443:Tyrosine; D004744:Enkephalin, Methionine