title: Isolation and characterization of R-phycoerythrin subunits and enzymatic digests.
abstract: Subunits and enzymatic digests of the highly fluorescent phycobiliprotein R-phycoerythrin (R-PE) were analyzed by several separation and detection techniques including HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), CE, and HPLC-electrospray ionization (ESI) MS. R-PE subunits were isolated by HPLC and detected as single molecules by total internal reflection fluorescence microscopy. The results show efficient absorption and fluorescence of the R-PE subunits and digest peptides, originating from the incorporation of phycoerythrobilin and phycourobilin chromophores in them. In addition, HPLC-ESI-MS and SDS-PAGE were optimized to determine the molecular masses of phycobiliprotein subunits and the chromophore-containing peptides, as well as the amino acid sequences of the latter. Favorable spectroscopic and structural properties of R-PE subunits and enzymatic digests, even under denaturing conditions, make these molecules suitable for use as fluorescence labels for biomolecules.
authors.affiliation: Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
authors.forename: Dragan
authors.initials: D
authors.lastname: Isailovic
authors.forename: Hung-Wing
authors.initials: HW
authors.lastname: Li
authors.forename: Edward S
authors.initials: ES
authors.lastname: Yeung
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010799:Phycoerythrin / Q000737:chemistry / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D010799:Phycoerythrin