Preparation and circular dichroism analysis of sodium hyaluronate oligosaccharides and chondroitin

Mary Cowman, Endre A. Balazs, Carl W. Bergmann, Karl Meyer

Research output: Contribution to journalArticle

Abstract

Sodium hyaluronate was cleaved into an homologous series of oligosaccharides by the action of bovine testicular hyaluronidase (EC 3.2.1.35), an endo-β-hexosaminidase. Digestion conditions and gel-filtration chromatographic fractionation were optimized to produce oligosaccharides, clearly separable into peaks corresponding to 1-23 disaccharide units of the type D-glucuronosyl-→-N-acetyl-D-glucosamine. The chromatographic method was also employed for the purification of a second homologous group of oligosaccharides, with the reversed sequence of monosaccharide units, produced by the action of leech hyaluronidase (EC 3.2.1.36), an endo-β-glucuronidase. Circular dichroism (CD) analysis (in the 200-250-nm range) of the oligosaccharides showed that the CD spectrum of hyaluronate in aqueous solution at neutral pH does not reflect to any substantial degree a polymer conformation which requires cooperative interaction between several repeating residues for stabilization. The enhanced CD properties of hyaluronate relative to those of monosaccharides are primarily related to the existence of the β-1,4 linkage from N-acetyl-D-glucosamine to D-glucuronate. Chondroitin, the N-acetyl-D-galactosamine analogue of hyaluronate, was prepared by chemical desulfation of chondroitin 4- and 6-sulfates. The purified product had a molecular weight range of 4000-8000 (10-20 disaccharide units). It was digested with testicular hyaluronidase, and the split products were isolated by gel filtration. In contrast to hyaluronate, the cleavage products included both the preponderant analogous repeating disaccharide multiples with N-acetyl-D-galactosamine at the reducing end and smaller quantities of oligosaccharides composed of an odd number of monosaccharides. These latter products were presumably derived from the ends of the shortened chondroitin chains, resulting from methanolysis during the desulfation and alkaline elimination of reducing hexosamines. CD spectroscopic analysis of chondroitin relative to its constituent monosaccharides showed that this glycosaminoglycan does not share the structural feature which results in substantially enhanced CD properties for hyaluronate. A hypothesis relating the CD properties of N-acetylated hexosamines in oligo- and polysaccharides to the dihedral angle about the carbon-oxygen bond at C3 may explain the CD dependence on both the hexosaminidic linkage and hexosamine configuration at C4.

Original languageEnglish (US)
Pages (from-to)1379-1385
Number of pages7
JournalBiochemistry
Volume20
Issue number5
StatePublished - 1981

Fingerprint

Chondroitin
Hyaluronic Acid
Circular Dichroism
Oligosaccharides
Monosaccharides
Hexosamines
Hyaluronoglucosaminidase
Disaccharides
Acetylgalactosamine
Acetylglucosamine
Chondroitin Sulfates
Gel Chromatography
Gels
Hexosaminidases
Glucuronic Acid
Spectroscopic analysis
Glucuronidase
Leeches
Dihedral angle
Fractionation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Preparation and circular dichroism analysis of sodium hyaluronate oligosaccharides and chondroitin. / Cowman, Mary; Balazs, Endre A.; Bergmann, Carl W.; Meyer, Karl.

In: Biochemistry, Vol. 20, No. 5, 1981, p. 1379-1385.

Research output: Contribution to journalArticle

Cowman, M, Balazs, EA, Bergmann, CW & Meyer, K 1981, 'Preparation and circular dichroism analysis of sodium hyaluronate oligosaccharides and chondroitin', Biochemistry, vol. 20, no. 5, pp. 1379-1385.
Cowman, Mary ; Balazs, Endre A. ; Bergmann, Carl W. ; Meyer, Karl. / Preparation and circular dichroism analysis of sodium hyaluronate oligosaccharides and chondroitin. In: Biochemistry. 1981 ; Vol. 20, No. 5. pp. 1379-1385.
@article{b3e3dae1b628488a8ebc88e352d961f1,
title = "Preparation and circular dichroism analysis of sodium hyaluronate oligosaccharides and chondroitin",
abstract = "Sodium hyaluronate was cleaved into an homologous series of oligosaccharides by the action of bovine testicular hyaluronidase (EC 3.2.1.35), an endo-β-hexosaminidase. Digestion conditions and gel-filtration chromatographic fractionation were optimized to produce oligosaccharides, clearly separable into peaks corresponding to 1-23 disaccharide units of the type D-glucuronosyl-→-N-acetyl-D-glucosamine. The chromatographic method was also employed for the purification of a second homologous group of oligosaccharides, with the reversed sequence of monosaccharide units, produced by the action of leech hyaluronidase (EC 3.2.1.36), an endo-β-glucuronidase. Circular dichroism (CD) analysis (in the 200-250-nm range) of the oligosaccharides showed that the CD spectrum of hyaluronate in aqueous solution at neutral pH does not reflect to any substantial degree a polymer conformation which requires cooperative interaction between several repeating residues for stabilization. The enhanced CD properties of hyaluronate relative to those of monosaccharides are primarily related to the existence of the β-1,4 linkage from N-acetyl-D-glucosamine to D-glucuronate. Chondroitin, the N-acetyl-D-galactosamine analogue of hyaluronate, was prepared by chemical desulfation of chondroitin 4- and 6-sulfates. The purified product had a molecular weight range of 4000-8000 (10-20 disaccharide units). It was digested with testicular hyaluronidase, and the split products were isolated by gel filtration. In contrast to hyaluronate, the cleavage products included both the preponderant analogous repeating disaccharide multiples with N-acetyl-D-galactosamine at the reducing end and smaller quantities of oligosaccharides composed of an odd number of monosaccharides. These latter products were presumably derived from the ends of the shortened chondroitin chains, resulting from methanolysis during the desulfation and alkaline elimination of reducing hexosamines. CD spectroscopic analysis of chondroitin relative to its constituent monosaccharides showed that this glycosaminoglycan does not share the structural feature which results in substantially enhanced CD properties for hyaluronate. A hypothesis relating the CD properties of N-acetylated hexosamines in oligo- and polysaccharides to the dihedral angle about the carbon-oxygen bond at C3 may explain the CD dependence on both the hexosaminidic linkage and hexosamine configuration at C4.",
author = "Mary Cowman and Balazs, {Endre A.} and Bergmann, {Carl W.} and Karl Meyer",
year = "1981",
language = "English (US)",
volume = "20",
pages = "1379--1385",
journal = "Biochemistry",
issn = "0006-2960",
number = "5",

}

TY - JOUR

T1 - Preparation and circular dichroism analysis of sodium hyaluronate oligosaccharides and chondroitin

AU - Cowman, Mary

AU - Balazs, Endre A.

AU - Bergmann, Carl W.

AU - Meyer, Karl

PY - 1981

Y1 - 1981

N2 - Sodium hyaluronate was cleaved into an homologous series of oligosaccharides by the action of bovine testicular hyaluronidase (EC 3.2.1.35), an endo-β-hexosaminidase. Digestion conditions and gel-filtration chromatographic fractionation were optimized to produce oligosaccharides, clearly separable into peaks corresponding to 1-23 disaccharide units of the type D-glucuronosyl-→-N-acetyl-D-glucosamine. The chromatographic method was also employed for the purification of a second homologous group of oligosaccharides, with the reversed sequence of monosaccharide units, produced by the action of leech hyaluronidase (EC 3.2.1.36), an endo-β-glucuronidase. Circular dichroism (CD) analysis (in the 200-250-nm range) of the oligosaccharides showed that the CD spectrum of hyaluronate in aqueous solution at neutral pH does not reflect to any substantial degree a polymer conformation which requires cooperative interaction between several repeating residues for stabilization. The enhanced CD properties of hyaluronate relative to those of monosaccharides are primarily related to the existence of the β-1,4 linkage from N-acetyl-D-glucosamine to D-glucuronate. Chondroitin, the N-acetyl-D-galactosamine analogue of hyaluronate, was prepared by chemical desulfation of chondroitin 4- and 6-sulfates. The purified product had a molecular weight range of 4000-8000 (10-20 disaccharide units). It was digested with testicular hyaluronidase, and the split products were isolated by gel filtration. In contrast to hyaluronate, the cleavage products included both the preponderant analogous repeating disaccharide multiples with N-acetyl-D-galactosamine at the reducing end and smaller quantities of oligosaccharides composed of an odd number of monosaccharides. These latter products were presumably derived from the ends of the shortened chondroitin chains, resulting from methanolysis during the desulfation and alkaline elimination of reducing hexosamines. CD spectroscopic analysis of chondroitin relative to its constituent monosaccharides showed that this glycosaminoglycan does not share the structural feature which results in substantially enhanced CD properties for hyaluronate. A hypothesis relating the CD properties of N-acetylated hexosamines in oligo- and polysaccharides to the dihedral angle about the carbon-oxygen bond at C3 may explain the CD dependence on both the hexosaminidic linkage and hexosamine configuration at C4.

AB - Sodium hyaluronate was cleaved into an homologous series of oligosaccharides by the action of bovine testicular hyaluronidase (EC 3.2.1.35), an endo-β-hexosaminidase. Digestion conditions and gel-filtration chromatographic fractionation were optimized to produce oligosaccharides, clearly separable into peaks corresponding to 1-23 disaccharide units of the type D-glucuronosyl-→-N-acetyl-D-glucosamine. The chromatographic method was also employed for the purification of a second homologous group of oligosaccharides, with the reversed sequence of monosaccharide units, produced by the action of leech hyaluronidase (EC 3.2.1.36), an endo-β-glucuronidase. Circular dichroism (CD) analysis (in the 200-250-nm range) of the oligosaccharides showed that the CD spectrum of hyaluronate in aqueous solution at neutral pH does not reflect to any substantial degree a polymer conformation which requires cooperative interaction between several repeating residues for stabilization. The enhanced CD properties of hyaluronate relative to those of monosaccharides are primarily related to the existence of the β-1,4 linkage from N-acetyl-D-glucosamine to D-glucuronate. Chondroitin, the N-acetyl-D-galactosamine analogue of hyaluronate, was prepared by chemical desulfation of chondroitin 4- and 6-sulfates. The purified product had a molecular weight range of 4000-8000 (10-20 disaccharide units). It was digested with testicular hyaluronidase, and the split products were isolated by gel filtration. In contrast to hyaluronate, the cleavage products included both the preponderant analogous repeating disaccharide multiples with N-acetyl-D-galactosamine at the reducing end and smaller quantities of oligosaccharides composed of an odd number of monosaccharides. These latter products were presumably derived from the ends of the shortened chondroitin chains, resulting from methanolysis during the desulfation and alkaline elimination of reducing hexosamines. CD spectroscopic analysis of chondroitin relative to its constituent monosaccharides showed that this glycosaminoglycan does not share the structural feature which results in substantially enhanced CD properties for hyaluronate. A hypothesis relating the CD properties of N-acetylated hexosamines in oligo- and polysaccharides to the dihedral angle about the carbon-oxygen bond at C3 may explain the CD dependence on both the hexosaminidic linkage and hexosamine configuration at C4.

UR - http://www.scopus.com/inward/record.url?scp=0019871867&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0019871867&partnerID=8YFLogxK

M3 - Article

VL - 20

SP - 1379

EP - 1385

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -