Analysis of Glycosaminoglycans with UNIT 17.13B Polysaccharide Lyases Polysaccharide lyases are a class of enzymes useful for analysis of glycosaminoglycans (GAGs) and the glycosaminoglycan component of proteoglycans (PGs). These enzymes cleave specific glycosidic linkages present in acidic polysaccharides and result in depo- lymerization (Linhardt et al., 1986). These enzymes act through an eliminase mechanism resulting in unsaturated oligosaccharide products that have UV absorbance at 232 nm. The lyases are derived from a wide variety of pathogenic and nonpathogenic bacteria and fungi (Linhardt et al., 1986). This class of enzymes includes heparin lyases (heparinases), heparan sulfate lyases (heparanases or heparitinases), chondroitin lyases (chondroiti- nases), and hyaluronate lyases (hyaluronidases), all of which are described in this unit. Polysaccharide lyases can be used, alone or in combinations, to confirm the presence of GAGs in a sample as well as to distinguish between different GAGs (see Table 17.13B.1 and Commentary). The protocols given for heparin lyase I are general and, with minor modifications (described for each lyase and summarized in Table 17.13B.2), can be used for any of the polysaccharide lyases. The basic protocol describes depolymerization of GAGs in samples containing 1 µg to 1 mg of GAGs. The alternate protocol describes depolymerization of GAGs in samples containing <1 µg of radiolabeled GAG. Two support protocols describe assays to confirm and quantitate the activity of heparin and chondroitin ABC lyases. It is recommended that enzyme activity be assayed before the enzyme is used in an experiment to be sure it is active and has been stored properly. The standard definition of a unit (U), 1 µmol product formed/min, is used throughout this µ ∆ article. Some lyases are sold in nonstandard units (e.g., 0.1 mol/hr, A232/min), and these should either be converted to standard units or the activity should be determined using the appropriate support protocol. Table 17.13B.1 Polysaccharide Lyases Used to Identify Glycosaminoglycans Glycosaminoglycan Enzyme Heparin, heparan sulfate, chondroitin sulfate, Heparin lyases I, II, III, and chondroitin dermatan sulfate, hyaluronate ABC, AC lyases Heparin, heparan sulfate, chondroitin sulfate, Heparin lyases I, II, III, and chondroitin dermatan sulfate ABC lyase Heparin, heparan sulfate Heparin lyases I, II, III Chondroitin sulfate, dermatan sulfate, Chondroitin ABC, AC lyases hyaluronate Chondroitin sulfate, dermatan sulfate Chondroitin ABC lyase Heparin (Heparin lyase I–heparin lyase III)a Heparan sulfate Heparin lyase III Chondroitin sulfate Chondroitin AC lyase (or chondroitin ABC lyase–chondroitin B lyase)a Dermatan sulfate Chondroitin B lyase (or chondroitin ABC lyase–chondroitin AC lyase)a Hyaluronate Hyaluronate lyase a Sample is divided into two portions and each is treated with a different lyase. The amount of depolymerization (i.e., Preparation and counts moving from Vo to Vt of a gel-filtration column) is determined for each portion, and the difference gives a measure Analysis of of the amount of glycosaminoglycan. Glycoconjugates Contributed by Robert J. Linhardt 17.13B.1 Current Protocols in Molecular Biology (1999) 17.13B.1-17.13B.16 Copyright © 1999 by John Wiley & Sons, Inc. Supplement 48 OVERVIEW OF HEPARIN LYASES There are three well-characterized polysaccharide lyases that act endolytically on heparin and heparan sulfate (Jandik et al., 1994); these enzymes are called heparin lyases. Heparin and heparan sulfate GAGs are structurally related, linear sulfated polysaccharides. Heparin’s major sequence (representing 70% to 90% of its structure) is as follows: →4)-α-D-glucosamine-2,6-sulfate-(1→4)-α-L-iduronic acid-2-sulfate-(1→ Heparan sulfate is composed primarily of equal proportions of the following: →4)-α-D-glucosamine-2-sulfate-(1→4)-β-D-glucuronic acid-(1→ and →4)-α-N-acetyl-D-glucosamine-6-sulfate-(1→4)-β-D-glucuronic acid (or α-L-iduronic acid)-(1→ These disaccharide sequences are found in differing amounts in both heparin and heparan sulfate (Desai et al., 1993a,b). Substrates for heparin lyases are illustrated in Figure 17.13B.1. The nomenclature of the three heparin lyases is somewhat confusing. However, heparin lyase I and III have enzyme commission (EC) numbers to facilitate their identification. The decision as to which enzyme should be used for a particular application is based on both the specificity desired and the reaction conditions required. The activity of these lyases toward specific glycosidic linkages has been determined using structurally char- acterized oligosaccharide substrates (Desai et al., 1993a). The primary linkages cleaved by these enzymes and their relative activities toward heparin and heparin sulfate (Desai et al., 1993a, b) are presented in Figure 17.13B.1 and Table 17.13B.3. Information on the optimal conditions for the activity and stability for these enzymes is given under the description of each enzyme and summarized in Table 17.13B.2. heparin lyase I heparin lyase II – CH2OX CH2OX CO2 O – O O O CO2 OX OH OX OH O O O O – – NHSO3 OSO3 NHY OX heparin lyase III – CH2OX CO2 O O OH OH O O NHY OH Analysis of Glycosamino- glycans with Polysaccharide Figure 17.13B.1 Primary glycosidic linkages cleaved by heparin lyases. Abbreviations: X, H or Lyases − − SO3 ; Y, CH3CO or SO3 . Heparin lyase II cleaves at either glucuronic or iduronic acid residues. 17.13B.2 Supplement 48 Current Protocols in Molecular Biology Table 17.13B.2 Reaction Conditions for Polysaccharide Lyases with Optimum Buffers and Reaction Temperatures Optimum Lyase Buffera temperature Heparin I Sodium phosphate/NaCl, pH 7.1 30°C Heparin II Sodium phosphate, pH 7.1 35°C Heparin III Sodium phosphate, pH 7.6 35°C Chondroitin ABC Tris⋅Cl/sodium acetate, pH 8 37°C Chondroitin AC Tris⋅Cl/sodium acetate, pH 8 37°C Chondroitin B Ethylenediamine/acetic 25°C acid/NaCl, pH 8 Hyaluronate Sodium acetate/NaCl, pH 5.2 >30°C a See Reagents and Solutions for buffer recipes. Table 17.13B.3 Activity of Heparin Lyases Activity and Heparin Heparin Heparin substrate conversion lyase I lyase II lyase III Heparina % Activityb 100 58 <1 % Conversionc 58 (76)d 85 6 Heparan sulfatee % Activity 13 100 100 % Conversion 19 39 94 aPorcine mucosal heparin. b Percent activity = [initial rate on the substrate examined/initial rate on substrate giving the highest activity] × (100). cPercent conversion = [moles of linkages cleaved/total moles of hexos- amine→uronic acid linkages] × (100). dBovine lung heparin. eBovine kidney heparan sulfate. HEPARIN LYASE I (Lohse and Linhardt, 1992) ENZYME Heparin lyase I (EC 4.2.2.7), from Flavobacterium heparinum (Cytophagia heparinia), is commonly referred to as heparinase. The enzyme has a molecular weight of 42,800 Da and a pI of 9.1 to 9.2. Heparin lyase I has a random endolytic action pattern—i.e., it randomly acts on any site with the appropriate primary structure within the polymeric substrate (Fig. 17.13B.1; Jandik et al., 1994). Complete Heparin Lyase–Catalyzed Depolymerization of an BASIC Unlabeled Sample PROTOCOL Samples consisting of tissues, biological fluids, PGs, and GAGs (UNIT 17.2) that contain microgram quantities of heparin and are not metabolically labeled can be analyzed using heparin lyase (see Critical Parameters for method to distinguish between heparin and heparan sulfate). Preparation and Analysis of Glycoconjugates 17.13B.3 Current Protocols in Molecular Biology Supplement 48 Materials Heparin- or heparan sulfate–containing sample Sodium phosphate/NaCl buffer (see recipe) Heparin lyase I solution (see recipe) Spectropor dialysis membrane, MWCO 1000 (Spectrum) 500-µl polypropylene microcentrifuge tubes 30° and 100°C water baths Additional reagents and equipment for polysaccharide dialysis (APPENDIX 3D), HPLC (UNIT 17.18), and gel-filtration chromatography (UNITS 10.9 & 17.17) 1. Dissolve sample, containing 1 µg to 1 mg heparin, in 50 µl sodium phosphate/NaCl buffer. Dialyze sample against sodium phosphate/NaCl buffer using 1000 MWCO dialysis membrane. 2. Thaw and assay activity of a frozen aliquot of enzyme. 3. Thaw 10 µl heparin lyase I solution at room temperature and add 40 µl sodium phosphate/NaCl buffer to the 500-µl tube containing enzyme. Add 50 µl sodium phosphate/NaCl buffer to another 500-µl tube to serve as a blank control. Additional enzyme (10- to 100-fold) may be required to break down small, resistant oligosaccharides (Rice and Linhardt, 1989; Desai et al., 1993a). For heparin lyase II and III, the chondroitin lyases, and hyaluronate lyase, see alternative conditions listed in Table 17.13B.2 and in descriptions of individual enzymes below. 4. Add 50 µl sample to each tube and incubate 8 to 12 hr at 30°C. 5. Heat tube 2 min at 100°C to terminate the reaction. Analyze the product by a method appropriate for its purity and concentration. A pure sample containing >10 ìg of heparin can be analyzed by measuring the difference ∆ in absorbance at 232 nm ( A232) in 30 mM HCl between enzyme-treated sample and blank (ε = 5500 M−1 for oligosaccharide products in 30 mM HCl). Enzyme-treated sample is diluted with measured amounts of 30 mM HCl until the A232 is between 1 and 2. The blank is diluted with the same quantity of 30 mM HCl and its A232 is measured. The difference ∆ between the two measured A232 values, A232, is used to calculate the moles of oligosac- charide product formed. Ten moles of oligosaccharide product is obtained for each mole of heparin. If the treated sample contains substantial amounts of protein or other sub- stances that absorb at or near 232 nm, disappearance of polysaccharide substrate can be measured using a dye-binding assay (Grant et al., 1984). Smaller quantities of samples or samples of lower purity can be analyzed by HPLC or gel-filtration chromatography using UV or conductivity detection methods.