An NMR study of 13C-enriched galactose attached to the single carbohydrate chain of hen ovalbumin. Motions of the carbohydrates of glycoproteins.
In order to facilitate the study of motions of the carbohydrate moiety of glycoproteins, a method of enzymatically attaching uniformly 13C-enriched galactopyranose ([U-13C]galactose) onto the nonreducing end of the carbohydrate chain has been developed. Specifically, [U-13C]glucose (20% 13C) was converted through a sequence of four enzymatic steps to [U-13C]UDP-galactose which was then attached, via a beta-1,4 glycosidic bond, to a nonreducing terminal N-acetylglucosamine of hen ovalbumin using the enzyme bovine galactosyltransferase. The overall reaction was followed using 31P and 13C NMR. 13C NMR spectra of the modified ovalbumin show six well resolved resonances arising from the six carbons of the attached [U-13C]galactose. Values of the spin-lattice relaxation time, the line width, the 13C (1H) nuclear Overhauser effect, and the intensity ratio, a measure of the off-resonance rotating frame spin-lattice relaxation time, were obtained for the individual carbons of the [U13-C]galactose as well as other carbon resonances of the protein. Relaxation data for the protein alpha-carbon and aromatic methine carbon resonances could be fit to theoretical values by assuming a correlation time of 25 ns for overall isotropic motion of the glycoprotein. Relaxation data for the anomeric carbon of the attached [U-13C]galactose could only be fit by assuming an additional correlation time between 40 and 80 ps for free internal motion, subject to the constraint that its carbon-proton bond vector makes an angle of about 30 degrees with an effective axis of rotation.
D000818:Animals; D002247:Carbon Isotopes; D002645:Chickens; D005690:Galactose / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010047:Ovalbumin / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D002247:Carbon Isotopes; D010047:Ovalbumin; D014532:Uridine Diphosphate Glucose; D005690:Galactose