Complete Heparin Lyase Catalyzed Depolymerization of Radiolabeled HS

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1. Dissolve GAG sample containing radiolabeled HS in 50 ^L of sodium phosphate buffer. Dialyze sample against sodium phosphate buffer using 1000 MWCO dialysis membrane or a Centricon (YM3, MWCO 3000) centrifugal filter unit (see Note 9).

2. Thaw 10 ^L of heparin lyase III solution at room temperature, immediately prior to use (see Note 5).

3. Add 30 ^L of sodium phosphate buffer to the 500-^L polypropylene microcentrifuge tube containing the enzyme solution.

4. (Optional) (see Note 15). Add 1.7 ^L each of 20-mg/mL chondroitin sulfate A, chon-droitin sulfate C, and dermatan sulfate substrate solutions (34 ^g of each GAG) to the enzyme in buffer.

5. Add 50 ^L of radiolabeled heparan sulfate solution and incubate 8-12 h at 30°C.

6. Heat at 100°C for 2 min to inactivate the enzyme.

7. Analyzed depolymerized radioactive sample by gel-permeation chromatography using radioisotope detection methods (see Note 14).

3.4. Analysis of Product

The heparin and heparan sulfate oligosaccharides can be analyzed by gradient PAGE, capillary electrophoresis (CE), or strong-anion-exchange HPLC (21-24).

4. Notes

1. Heparin lyase I from Flavobacterium heparinum is sold as heparinase I by Sigma and as heparinase by Seikagaku. Heparin lyase II is sold as heparinase II by Sigma and as heparitinase II by Seikagaku. Heparin lyase III from Flavobacterium heparinum is sold as heparinase III by Sigma and as heparatinase or heparatinase I by Seikagaku.

2. Often the purchased lyophilized enzyme contains bovine serum albumin (BSA) as a stabilizer. For example, Sigma samples contain 25% BSA.

3. Samples consisting of tissue, biological fluids, PGs, and GAGs that contain microgram quantities of HS can often be analyzed directly using heparin lyases without the use of radioisotopes.

4. Since calcium is an activator for heparin lyase I and III, 20 mM sodium acetate buffer in the presence of 2 mM calcium acetate can be used with these enzymes. These enzymes are also compatible with a wide range of other biological buffers.

5. Storage: The lyophilized enzyme is stable at -20°C for at least 2 yr. The dissolved enzyme is stable when frozen at -20°C for 1 mo and for over a year at -70°C. The heparin lyases are sensitive to freeze-thawing, especially heparin lyase III. Once heparin lyase III samples are thawed, they should be used immediately.

6. If a temperature-controlled spectrophotometer is not available, activity can be measured at room temperature, or samples can be incubated in a water bath and the absorbance can be measured at fixed time points.

7. One unit is equal to 1 ^mol product formed per minute.

8. If the sample is believed to contain HS, heparin lyase III should be used. Samples that contain heparin should be treated with heparin lyase I. In samples where the identity of the GAG is unknown or believed to be a mixture of HS and heparin use either heparin lyase II or an equal unit mixture of heparin lyase I, II, and III to ensure complete depoly-merization.

9. The presence of metals, detergents, and denaturants can interfere with the activity of the lyases. Before digesting the samples, detergents should be removed by precipitation with potassium chloride or by using a detergent-removal column such as Biobeads (Bio-Rad). Urea and guanidine should be removed by exhaustive dialysis using controlled-pore dialysis membrane (MWCO 1000).

10. Additional enzyme (10- to 100-fold) may be required to break down small, resistant oligosaccharides (25-26).

11. If possible, gently shake the samples during digestion.

12. Following the use of a lyase, residual lyase activity can be destroyed by heating the reaction mixture to 100°C or by adding denaturants or detergents. Most lyases are cationic proteins and can be removed from anionic oligosaccharide products by passing the reaction mixture through a small cation-exchange column, such as SP-Sephadex (Sigma), adjusted to an acidic pH. The oligosaccharide products (void volume) are then recovered, readjusted to neutral pH, and analyzed. This method can also be used to remove BSA, an excipient found in many of the commercial enzymes, from the oligosaccharide products.

13. This assay is to be used with relatively pure GAGs. High concentrations of protein interfere with the measure of oligosaccharide production due to UV absorbance of the protein.

14. In gel-permeation chromotography of HS/heparin, following complete depolymerization using the appropriate heparin lyase, the products should elute close to the column's total volume, corresponding to an apparent molecular weight <1500 daltons (confirming the presence of heparin/HS), while the substrate (control without enzyme) should elute close to the column's void volume, corresponding to a molecular weight of >10,000 daltons.

15. When attempting to use heparin lyases to depolymerize radiolabeled samples that contain very small quantities of heparin or heparan sulfate, it is often useful to add cold substrate as a carrier so that the activity of heparin lyase can be distinguished from that of trace amounts of chondrotin lyases that may be present in heparin lyase preparations (27).


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