A new fluorogenic assay for tyrosine-containing peptides.
A new tyrosine-specific LC assay with pre-column fluorogenic derivatization is described for Tyr-Gly as model peptide. o-Hydroxylation of the tyrosine residue with tyrosinase in the presence of ascorbic acid, followed by oxidation to the corresponding quinone by potassium ferricyanide at room temperature and condensation with 1,2-diamino-1,2-diphenylethane in the presence of acetonitrile gave a highly fluorescent species. The resulting fluorescence signal was stable over the investigated period of 5 h and exhibited a linear response curve on a reversed-phase LC system. Under optimized reaction conditions, the lower limit of detection for Tyr-Gly was 200 fmol per injection. Examination of a series of dipeptides (L-Tyr-L-X; X = Gly, Ala, Val, Leu, Phe) showed no significant influence of neighbouring amino acids on the enzymatic hydroxylation by tyrosinase. This and the formation of a highly fluorescent signal for Leu-enkephalin suggests the general feasibility of the approach for the determination of tyrosine-containing peptides.
Tellier
M
M
College of Pharmacy, University of Florida, Gainesville 32610.
Prankerd
R J
RJ
Hochhaus
G
G
D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004151:Dipeptides / Q000032:analysis* / Q000378:metabolism; D005029:Ethylenediamines; D005292:Ferricyanides / Q000737:chemistry; D005453:Fluorescence; D006900:Hydroxylation; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D014443:Tyrosine / Q000032:analysis* / Q000737:chemistry
D004151:Dipeptides; D005029:Ethylenediamines; D005292:Ferricyanides; D010455:Peptides; D014443:Tyrosine; C009870:meso-1,2-diphenylethylenediamine; D014442:Monophenol Monooxygenase; C028033:potassium ferricyanide