Conformational studies of glycopeptides by energy transfer. Introduction of fluorophore at specific branches of biantennary glycopeptides.
Biantennary glycopeptides from bovine fibrinogen were fluorescence labeled at each branch specifically for conformational studies by fluorescence energy transfer. Glycopeptides (by Pronase digestion) were separated by anion-exchange chromatography based on the degree of sialylation. The major monosialyl biantennary glycopeptides (see below) were used as substrates for galactose oxidase and periodate oxidation. [Formula: See Text] Galactose oxidase was used to oxidize the terminal Gal6' located on the Man alpha(1-6)Man branch. The oxidized glycopeptides (containing 6-oxo-galactose) were modified with 2-(dansylamido)ethylamine by reductive amination. The N terminus of the peptide portion was then modified with naphthylacetic acid. Alternatively, the peptide portion of the monosialylated glycopeptide was first modified with naphthylacetic acid and the sialic acid located on the Man alpha(1-3)Man branch was oxidized with periodate under controlled conditions. The oxidized glycopeptides (oxo-sialic acid) were coupled with 2-(dansylamido)ethylamine by reductive amination. These doubly fluorescence-labeled glycopeptides were used for conformational studies of biantennary glycopeptides by energy transfer (see the accompanying article (Wu, P., Lee, K. B., Lee, Y. C., and Brand, L. (1996) J. Biol. Chem. 271, 1470-1474). Furthermore, the unmodified branch of the fluorescent labeled glycopeptides were digested stepwise with exoglycosidases. Resonance energy transfer experiments were done with each of the resulting derivatives to determine the effects of removing sugars at each stage of peeling on any conformational change on the resulting branch antennae.
Lee
K B
KB
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Lee
Y C
YC
D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D005340:Fibrinogen / Q000737:chemistry*; D005456:Fluorescent Dyes; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D007202:Indicators and Reagents; D007668:Kidney / Q000201:enzymology; D008297:Male; D008358:Mannose; D008969:Molecular Sequence Data; D011402:Pronase; D013737:Testis / Q000201:enzymology
D002241:Carbohydrates; D005456:Fluorescent Dyes; D006020:Glycopeptides; D007202:Indicators and Reagents; D005340:Fibrinogen; D006026:Glycoside Hydrolases; D011402:Pronase; D008358:Mannose