NMR methods to monitor the enzymatic depolymerization of heparin.
Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used (1)H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the (1)H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion.
Limtiaco
John F K
JF
University of California, Riverside, CA 92521, USA.
Beni
Szabolcs
S
Jones
Christopher J
CJ
Langeslay
Derek J
DJ
Larive
Cynthia K
CK
D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D019763:Heparin Lyase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009844:Oligosaccharides / Q000378:metabolism; D013552:Swine; D014466:Ultraviolet Rays
D000925:Anticoagulants; D009844:Oligosaccharides; D006493:Heparin; D019763:Heparin Lyase
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