Chemical change involved in the oxidative reductive depolymerization of hyaluronic acid.
The oxidative reductive depolymerization (ORD) of hyaluronate has been investigated. A solution of hyaluronate (Mr 4.07 x 10(5] in phosphate buffer (pH 7.2) was incubated in the presence of Fe2+ for 24 h at 37 degrees C under an oxygen atmosphere to yield depolymerized hyaluronate (ORD fragments; an average Mr of 2,600). The ORD fragments contain 21 and 24% less hexosamine and uronic acid, respectively, but no olefinic linkage. They were exhaustively digested with chondroitinase AC-II. The resulting oligosaccharides and monosaccharides were separated by gel filtration and ion-exchange chromatography, and their structures were determined by proton and carbon-13 NMR, fast atom bombardment mass spectrometry, and chromatographic techniques combined with chemical modifications. The following structures derived from the reducing ends of the ORD fragments were identified: 4,5-unsaturated GlcA(beta 1----3)-N-acetyl-D-glucosaminic acid (where GlcA- represents glucuronosyl-) (21%), 4,5-unsaturated GlcA(beta 1----3)GlcNAc(beta 1----3)-D-arabo-pentauronic acid (24%), and N-acetyl-D-glucosamine (51%). The following structures derived from the nonreducing ends were identified: L-threo-tetro-dialdosyl-(1----3)GlcNAc (a tentative structure, 8%), N-acetylhyalobiuronic acid (20%), and N-acetyl-D-glucosamine (45%). The results indicate that the ORD reaction of hyaluronate proceeds essentially by random destruction of unit monosaccharides due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable glycosidic substituents.
Uchiyama
H
H
School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
Dobashi
Y
Y
Ohkouchi
K
K
Nagasawa
K
K
D000117:Acetylglucosamine / Q000032:analysis; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000302:isolation & purification; D055598:Chemical Phenomena; D002621:Chemistry; D002808:Chondroitin Lyases / Q000378:metabolism; D002845:Chromatography; D005965:Glucuronates / Q000032:analysis; D020723:Glucuronic Acid; D006820:Hyaluronic Acid / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D011108:Polymers; D012996:Solutions
D002241:Carbohydrates; D005965:Glucuronates; D009844:Oligosaccharides; D011108:Polymers; D012996:Solutions; D020723:Glucuronic Acid; D006820:Hyaluronic Acid; D002808:Chondroitin Lyases; D000117:Acetylglucosamine