Carbohydrate Analysis III

Complex Carbohydrates

Heparinase, Heparin and Heparan Sulfate

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 No. 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 No. 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 No. H8891

Hyaluronidase and Hyaluronic Acid

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 we 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. We have 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 ours 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 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 No. H3631 (3,000-15,000 un/mg)
Product No. H3884 (750-1500 un/mg)
Product No. H4272 (750-1500 un/mg, embryo tested)
Product No. H3506 (300-750 un/mg)
Product No. H3757 (~300 un/mg)

from sheep testes
Product No. H6254 (minimum 1500 un/mg)
Product No. 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 No. H1136

Inulinase and Inulin

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

Pectinases and Pectins

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 No. P2611 (Novozymes Pectinex Ultra SPL)

from Aspergillus niger
Product No. P4716 (aqueous glycerol solution, min. 5 un/mg protein)

from Rhizopus species
Product No. P2401 (Crude Powder 400-800 un/g)

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 No. P3026 (lyophilized, min. 0.3 un/mg)
Product No. P5936 (lyophilized, min. 2 un/mg)

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 No. P5400 (lyophilized, 350-700 un/mg)

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.

Lysozyme and Peptidoglycans

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 No. L6876 (lyophilized, approx 50,000 un/mg)
Product No. L7773 (Asceptically filled, lyophilized, approx 50,000 un/mg)
Product No. L2879 (chloride, lyophilized, approx 60,000 un/mg)
Product No. L0289 (biotin-caproyl, lyophilized, > 20,000 un/mg)

from human milk
Product No. L1667 (recombinant, expressed in rice, lyophilized, minimum 100,000 un/mg)

from human neutrophils
Product No. L8402 (lyophilized, minimum 100,000 un/mg)