Abstract
13C NMR spectroscopy has been used to characterize Amadori (ketoamine) adducts formed by reaction of [2-13C]glucose with free amino groups of protein. The spectra of glycated proteins were acquired in phosphate buffer at pH 7.4 and were interpreted by reference to the spectra of model compounds, N alpha-formyl-N epsilon-fructose-lysine and glycated poly-L-lysine (GlcPLL). The anomeric carbon region of the spectrum (approximately 90-105 ppm) of glycated cytochrome c was superimposable on that of N alpha-formyl-N epsilon-fructose-lysine, and contained three peaks characteristic of the alpha- and beta-furanose and beta-pyranose anomers of Amadori adducts to peripheral lysine residues on protein (pK alpha approximately 10.5). The spectrum of GlcPLL yielded six anomeric carbon resonances; the second set of three was displaced about 2 ppm to lower shielding of the first and was assigned to the Amadori adduct at the alpha-amino terminus (pK alpha approximately 7.5). The spectrum of glycated RNase was similar to that of GlcPLL, but contained a third set of three signals attributable to modification of active site lysine 41 (pK alpha approximately 8.8). The assignments for RNase were confirmed by analysis of spectra taken at pH 4 and under denaturing conditions. The spectrum of glycated hemoglobin was comparable to that of GlcPLL, and distinct resonances could be assigned to Amadori adducts at amino-terminal valine and intrachain N epsilon-lysine residues. Chemical analyses were performed to measure the relative extent of alpha- and epsilon-amino group modification in the glycated macromolecules, and the results were compared with estimates based on integration of the NMR spectra.
Highlights
From the $Department of Chemistry and §School of Medicine, University of South Carolina, Columbia, South Carolina 29208
Increased glycation and ize Amadori adducts formed by reaction subsequent Maillard reactions of protein are thought to be of [2-'sC]glucose with free amino groups of protein. involved in the structural and functional changes in body
The spectrumof GlcPLL yielded six furanose conformations in solution [7], relatively little is known about the distribution of structures which exists on protein either in uiuoor in vitro
Summary
Published in Journal of Biological Chemistry, Volume 260, Issue 9, 1984, pages 5406-5410. This research was originally published in the Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology. THE JOURNOAFLBIOLOGICACLHEMIsTRY Q 1985 by The American Society of Biological Chemists, Inc. Vol 260, No 9, Issue of May 10, pp, 54"5410,1986 Printed in U.S.A
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.