Enzyme Preparation and Storage

1. Tris-HCl/sodium acetate buffer, 50 mM (see Table 2 for pH).

Table 2

Properties of the Chondroitinase Lyases

Chondroitin Lyase/Organism MW (Da)

Buffer system

Chondroitinase ABC from Proteus vulgaris Chondroitinase ACI from Flavobacterium heparinum Chondroitinase ACII from Arthrobacter aurescens Chondroitinase B from Flavobacterium heparinum Chondroitinase C from Flavobacterium heparinum

150,000 Tris-HCl/sodium acetate 8.0 37

76,000 Tris-HCl/sodium acetate 7.5 37

76,000 Tris-HCl/sodium acetate 6.0 37

55,000 Tris-HCl/sodium acetate 7.5 25

— Tris-HCl/sodium acetate 8.0 25

2. Chondroitin lyase. The decision of which lyase to use should be based on the specificity desired (see Fig. 1 and Table 1).

3. 500-^L polypropylene microcentrifuge tubes.

2.2. Sample Preparation and Enzymatic Digestion

1. Tris-HCl/sodium acetate buffer, 50 mM (for pH see Table 2).

2. Chondroitin lyase solution.

3. CS- or DS-containing sample.

4. Spectropor dialysis tubing (1000-MWOT Spectrum or Centricon (YM3, & MWCO 3000, Millipore) centrifugal filter unit.

5. 500-^L polypropylene microcentrifuge tubes.

2.3. Assay Protocol

1. Tris-HCl/sodium acetate buffer, 50 mM (see Table 2 for pH).

2. Chondroitin lyase solution.

3. CS- or DS-containing sample.

4. 500-mL polypropylene microcentrifuge tubes.

5. UV-spectrophotometer, temperature controlled.

6. 1-mL quartz cuvet.

7. Radiolabel-containing sample.

8. Dialysis membrane (MWCO 1000) or Centricon (YM3, MWCO 3000) centrifugal filter unit.

9. 500-^L polypropylene microcentrifuge tubes.

10. Water baths at 30° and 35°C for enzyme digestion and at 100°C for inactivation of the enzyme reaction.

11. Additional reagents and equipment for product analysis, such as, SAX-HPLC, GPC, PAGE, CE, MS, and NMR.

3. Methods

3.1. Enzyme Preparation and Storage

1. Dissolve the commercial enzyme (see Note 1) by adding buffer directly to each vial to afford a 4 mU/mL final concentration (see Note 2). Cap the vials tightly and gently agitate until the solids are completely dissolved.

2. Dispense 10-^L aliquots of the enzyme solution for storage.

3. Store tubes containing enzyme at -60 to -80°C (see Note 3).

3.2. Sample Preparation and Enzymatic Digestion

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