Carbohydrate Analysis

Heparin and Heparan Sulfate
These glycosaminoglycans (GAGs) are complex heterogeneous mixtures of repeating disaccharide units consisting of a uronic acid (D-glucuronic or L-iduronic acid) and D-glucosamine or N-acetyl-D-glucosamine. Various degrees of sulfation occur (at O and/or N) on each monosacchiride unit, ranging from zero to tri-sulfation. In general, heparan is less sulfated than heparin.

Heparinase Specificities
Heparinase selectively cleaves sulfated glycans containing α(1-4) glycosidic linkages between the glucosamine and uronic acid residues in the heparin polymer. The cleavage proceeds via an elimination reaction, resulting in the formation of oligosaccharides containing unsaturated uronic acid residues (double bond between C4 and C5). These cleavage products can be detected by UV spectroscopy (232 nm).

The three forms of heparinase (I, II, and III) have varying substrate specificities, as follows:

Heparinase I
(Heparin Lyase I)
EC# 4.2.2.7

Heparinase I cleaves heparin and heparan sulfate (relative activity about 3:1) at the linkages between hexosamines and O-sulfated iduronic acids, yielding mainly disaccharides. The enzyme also cleaves the antithrombin III binding pentasaccharide domain in the heparin molecule.

from Flavobacterium heparinum
Product Number H2519

Heparinase II
(Heparin Lyase II)

Heparinase II cleaves heparan sulfate, and to a lesser extent heparin (relative activity about 2:1), at the 1-4 linkages between hexosamines and uronic acid residues (both glucuronic and iduronic), yielding mainly disaccharides.
The lyase activity of Heparinase II had been previously characterized as 'heparitinase II' by Silva, M.E., and Dietrich, C.P.

from Flavobacterium heparinum
Product Number H6512

Heparinase III
Heparinase III cleaves at the 1-4 linkages between hexosamine and glucuronic acid residues in heparan sulfate, yielding mainly disaccharides. The enzyme is not active towards heparin or low molecular weight heparins.
The lyase activity of Heparinase III had been previously characterized as 'heparitinase I' by Silva, M.E., and Dietrich, C.P.

from Flavobacterium heparinum
Product Number H8891

References
1. Ernst, S., et al., Enzymatic Degradation of Glycosaminoglycans. Critical Reviews in Biochemistry and Molecular Biology, 30 (5), 387-444 (1995).
2. Desai, U.R.,Wang, H., and Linhardt, R.J., Substrate Specificity of the Heparin Lyases from Flavobacterium heparinum. Archives of Biochemistry and Biophysics, 306(2), 461-468 (1993).
3. Schultz, J. S., Biotechnol. Prog., 3, 27-30 (1987).
4. Nader, H. B., et al., Purification and Substrate Specificity of Heparitinase I and Heparitinase II from Flavobacterium Heparinum. Analyses of the Heparin and Heparan Sulfate Degradation Products by ¹³C NMR Spectroscopy. J. Biol. Chem., 265(28), 16807-16813 (1990).
5. Nader, H. B., et al., Heparin Sequences in the Heparan Sulfate Chains of an Endothelial Cell Proteoglycan. Proc. Natl. Acad. Sci. USA, 84, 3565-3569 (1987).
6. Hovingh, P., and Linker, A., The Disaccharide Repeating-units of Heparan Sulfate. Carbohydr. Res., 37, 181-192 (1974).
7. Linker, A., and Hovingh, P., Heparinase and Heparitinase from Flavobacteria. Meth. Enzymol., 28-B, 902-911 (1972).
8. Silverberg, I., et al., Carbohyd. Res., 137, 227 (1985).
9. Ototani, N., et al., Purification of Heparinase and Heparitinase by Affinity Chromatography on Glycosaminoglycan-bound AH-Sepharose 4B. Carbohydr. Res., 88, 291-303 (1981).
10. Lingardt, R.J., et al., Mode of action of heparin lyase on heparin. Biochem. Biophys. Acta., 702(2), 197-203 (1982).
11. Linker, A., et al., Heparinase and Heparitinase from Flavobacteria. Methods of Enzymology, 28, 902-911 (1972).
12. Lingardt, R.J., et al., Examination of the substrate specificity of heparin and heparan sulfate lyases. Biochem., 29(10), 2611-2617 (1990).
13. Proc. 8th Int. Symposium Glycoconjugates, 1, 73 (1985).
14. Silva, M.E., and Dietrich, C.P., Isolation and partial characterization of three induced enzymes from Flavobacterium heparinum involved in the degradation of heparin and heparitin sulfates., Biochem. Biophys. Res. Commun., 56, 965–972 (1974)

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Composed of alternating residues of β-D-(1-3) glucuronic acid and β-D-(1-4)-N-Acetylglucosamine

Specificity
The mammalian hyaluronidases (EC# 3.2.1.35) cleave hyaluronic acid and similar glycosaminoglycans by hydrolysis. The enzyme from Streptomyces (EC# 4.2.2.1) is a lyase that catalyzes cleavage by an elimination reaction yielding a 4-deoxy-4,5-unsaturated oligosaccharides. It’s specificity towards chondroitins and other glycosaminoglycans is unclear.

Hyaluronidase Assays
Prior to 2008 Sigma-Aldrich utilized the Hyaluronidase assay procedure described in the USP (United States Pharmacopoeia XXIII-National Formulary XVIII Combined Edition). The unit activity was defined using a USP standardized hyaluronidase enzyme. This unit was defined as: One unit is based on the change in absorbance at 600 nm (change in turbidity) of a USP reference standard hyaluronidase which is assayed concurrently with each lot of this product.

The USP no longer offers a standardized hyaluronidase enzyme. Sigma-Aldrich has modified the assay method and unit definition to accommodate this unavoidable change. The new unit definition is: One unit will cause a change in A600nm of 0.330 per minute at pH 5.7 at 37 ºC (45 minute assay).

The change in absorbance value of 0.330 in the new unit definition was chosen in order to most closely match the results found using the USP hyaluronidase standard defined activity. As a result, the discontinued USP-based unit definition and the new unit Sigma-Aldrich unit definition will give a conversion factor of approximately 1:1 (One old unit will equal approximately one new unit). 

Assay Procedures
Original USP-Based Procedure
Current Sigma-Aldrich Method

Enzyme Products

Hyaluronidase
EC# 3.2.1.35
Synonyms: Hyaluronoglucosaminidase

The mammalian glycolytic hyaluronidase (EC# 3.2.1.35 ) catalyzes the random hydrolysis of the 1-4 bond between N-acetyl-D-glucosamine and D-glucuronic acid in hyaluronic acid. It also hydrolyses 1,4-beta-D-glycosidic linkages between N-acetyl-galactosamine or N-acetylgalactosamine sulfate and glucuronic acid in chondroitin sulfates A and C , and dermatan.

from bovine testes 
Product Number H3631 (3,000-15,000 un/mg)
Product Number H3884 (750-1500 un/mg)
Product Number H4272 (750-1500 un/mg, embryo tested)
Product Number H3506 (300-750 un/mg)
Product Number H3757 (~300 un/mg)

from sheep testes 
Product Number H6254 (minimum 1500 un/mg)
Product Number H2251 (minimum 500 un/mg)
Product Number H2126 (minimum 300 un/mg)

Hyaluronidase (Hyaluronate Lyase)
EC# 4.2.2.1
Synonyms: Hyaluronate Lyase

Cleaves hyaluronic acid at the β-D-GalNAc-(1-4)-β-D-GlcA bond, ultimately breaking the polysaccharide down to 3-(4-deoxy-β-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine

from Streptomyces hyaluronolyticus
Product Number H1136

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Inulin
Inulins are fructan oligosaccharides composed α-D-glucopyranosyl-[β-(2-1)D-fructofuranosyl-D-fructofuranosides. Inulins can generally contain 2 to 140 fructose units.

Inulinase
EC# 3.2.1.7
Synonyms: endo-1,4-β-xylanase

Inulinase catalyzes endohydrolysis of 2,1-β-D-fructosidic linkages in inulin

from Aspergillus niger
Product Number I2017 (Novozymes Fructozyme L)

References
1. Enzyme Nomenclature, (www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/8.html)
2. Azhari, R., et al., Biotechnol. Appl. Biochem. 11, 105-117 (1989)

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Pectin
Pectins are complex branched heteropolysaccharides primarily containing an α-(1-4) polygalacturonic acid backbone which can be randomly acetylated and methylated. Three different pectins have been isolated from plant cell walls.

• Homogalacturonans are composed of the simple α-(1-4) polygalacturonic acid backbone.
• Substituted homogalacturonans are modifications of this backbone with β-D-xylose branching at C3, or apiofuranose substitutions in the backbone with β-D-Apiosyl-(1,3')-β-D-Apiose branching.
• Rhamnogalacturonan I contains alternating α-(1-4) galacturonosyl and α-(1-2) rhamnosyl residues, with primarily oligo α-(1-3) arabinose and oligo β-(1-4) galactose branching.
• Rhamnogalacturonan II is composed of the simple α-(1-4) polygalacturonic acid backbone with complex branching with composed of up to 11 different monosaccharide types.

Pectinase
EC# 3.2.1.15
Synonyms: polygalacturonase

Pectinase catalyzes the random hydrolysis of 1,4-α-D-galactosiduronic linkages in pectin and other galacturonans.

from Aspergillus aculeatus
Product Number P2611 (Novozymes Pectinex Ultra SPL)

from Aspergillus niger
Product Number P2736 (Novozymes Pectinex 3XL)
Product Number P4716 (aqueous glycerol solution, min. 5 un/mg protein)
Product Number P0690 (plant cell culture tested)

from Rhizopus species
Product Number P2401 (Crude Powder 400-800 un/g)
Product Number P4300 (plant cell culture tested)

References
1. Enzyme Nomenclature (www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/15)

Pectolyase
EC# 3.2.1.15 & 4.2.2.10
Reported to contain two types of pectinase, endopolygalacturonase (EC3.2.1.15), endo-pectin lyase (EC4.2.2.10) and a maceration stimulating factor.

Pectolyase catalyzes the eliminative cleavage of (1 4)-α-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl- α-D-galact-4-enuronosyl groups at their non-reducing ends. Pectinase catalyzes the random hydrolysis of 1,4-α-D-galactosiduronic linkages in pectin and other galacturonans.

from Aspergillus japonicus
Product Number P3026 (lyophilized, min. 0.3 un/mg)
Product Number P5936 (lyophilized, min. 2 un/mg)
Product Number P5431 (plant cell culture tested)

References
1. Enzyme Nomenclature (www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/15 & www.chem.qmul.ac.uk/iubmb/enzyme/EC4/2/2/10)

Pectinesterase
EC# 3.1.1.11
Synonyms: pectin methylesterase

Pectinesterase catalyzes the hydrolysis of the methyl esters of pectin to yield pectate and methanol.

from orange peel
Product Number P5400 (lyophilized, 350-700 un/mg)
Product Number P0764 (lyophilized, 50-350 un/mg)

References
1. http://www.biobase.de/brenda/php/result_flat.php4?ecno=3.1.1.11

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Pullulanase

Pullulanase
EC# 3.2.1.41
Synonyms: Amylopectin 6-gluconohydrolase, Limit dextrinase

Pullulanase catalyzes the hydrolysis of (1-6)-α-D-glucosidic linkages in pullulan (a linear polymer of α-(1-6)-linked maltotriose units), and, similar to isoamylase, in amylopectin and glycogen, and the α- and β-limit dextrins of amylopectin and glycogen.

from Bacillus acidopullulyticus
Product Number P2986 (solution, min 10 Novozymes un/g, Novozymes Promozyme 400L)

from Klebsiella pneumoniae
Product Number P1067 (10-30 un/mg protein, lyophilized powder)
Product Number P5420 (≥5 un/mg protein, ammonium sulfate suspension)

References
1. Enzyme Nomenclature www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/41.html

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Polymer of β-(1-4)-N-Acetyl-D-glucosamine units. Alternating residues are modified to form N-acetylmuramic acid with the addition of lactate to form branching links to the tetrapeptide.

Lysozyme
EC# 3.2.1.17
Synonyms: peptidoglycan N-acetylmuramoylhydrolase

Lysozyme catalyzes the hydrolysis of 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins

from chicken egg white
Product Number L6876 (lyophilized, approx 50,000 un/mg)
Product Number L7651 (molecular biology grade, lyophilized, approx 50,000 un/mg)
Product Number L7001 (lyophilized, approx 50,000 un/mg)
Product Number L7773 (Asceptically filled, lyophilized, approx 50,000 un/mg)
Product Number L2879 (chloride, lyophilized, approx 60,000 un/mg)
Product Number L0289 (biotin-caproyl, lyophilized, > 20,000 un/mg)

from human milk
Product Number L6394 (lyophilized, minimum 100,000 un/mg)
Product Number L1667 (recombinant, expressed in rice, lyophilized, minimum 100,000 un/mg)

from human neutrophils
Product Number L8402 (lyophilized, minimum 100,000 un/mg)

References
1. Enzyme Nomenclature (www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/17.html)

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