Chemical change involved in the oxidative reductive depolymerization of hyaluronic acid

• Published in: The Journal of biological chemistry 1990-05, Vol.265 (14), p.7753-7759
• Main Authors: UCHIYAMA, H, DOBASHI, Y, OHKOUCHI, K, NAGASAWA, K
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - analysis; Biological and medical sciences; Carbohydrate Metabolism; Carbohydrate Sequence; Carbohydrates - isolation & purification; Chemical Phenomena; Chemistry; Chondroitin Lyases - metabolism; Chromatography; Fundamental and applied biological sciences. Psychology; Glucuronates - analysis; Glucuronic Acid; Hyaluronic Acid - metabolism; Intermolecular phenomena; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Miscellaneous; Molecular biophysics; Molecular Sequence Data; Molecular Structure; Molecular Weight; Oligosaccharides - isolation & purification; Oligosaccharides - metabolism; Oxidation-Reduction; Polymers; Solutions
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)38993-8

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.