Protein Protocols Protein Protocols

898 Turnbull
Table 1
Exoenzymes for Sequencing Heparan Sulfate and Heparin
Enzyme a Substrate specificity b
Sulfatases
Iduronate-2-sulfatase IdoA(2S)
Glucosamine-6-sulfatase GlcNAc(6S), GlcNSO 3(6S)
Sulphamidase (glucosamine N-sulfatase) GlcNSO 3
Glucuronate-2-sulfatase GlcA(2S)
Glucosamine-3-sulfatase GlcNSO 3(3S)
Glycosidases
Iduronidase IdoA
Glucuronidase GlcA
α-N-Acetylglucosaminidase GlcNAc
Bacterial exoenzymes
∆4,5-Glycuronate-2-sulfatase ∆UA(2S)
∆4,5-Glycuronidase ∆UA
a Enzyme availability: Glucuronidase is widely available commercially as purified or recombinant
enzyme. Recombinant iduronate-2-sulfatase, iduronidase, glucosamine-6-sulfatase, sulfamidase, and
α-N-acetylglucosaminidase are available from Glyko (Novato, CA; www.glyko.com). Glucuronate-
2-sulfatase and glucosamine-3-sulfatase have only been purified from cell and tissue sources to date. The
bacterial exoenzymes are available from Grampian Enzymes, Nisthouse, Harray, Orkney, Scotland;
e-mail, grampenz@aol.com.
b The specificities are shown as the nonreducing terminal group recognized by the enzymes. Sulfatases
remove only the sulfate group, whereas the glycosidases cleave the whole nonsulfated monosaccharide.
1. Dissolve the sample (typically 10–200 pmol of saccharide) in 10 µL of H 2O in a
microcentrifuge tube.
2. Add 5 µL of exoenzyme buffer (100 mM sodium acetate buffer, pH 4.5), 1 µL of 0.5 mg/mL
bovine serum albumin, 2 µL of appropriate exoenzyme [0.2–0.5 mU], and distilled water
to bring the final volume to 20 µL.
3. Mix the contents well on a vortex mixer, and centrifuge briefly to ensure that the reactants
are at the tip of the tube.
4. Incubate the samples at 37°C for 16 h in a heating block or oven.
3.4. Separation of Saccharides by PAGE
PAGE is a high-resolution technique for the separation of HS and heparin saccharides
of variable sulfate content and disposition. Its resolution is generally superior to
gel filtration or anion-exchange HPLC (19,20). Improved resolution can be obtained
using gradient gels, although these are more difficult to prepare and use routinely. In
most cases sufficient resolution can be obtained with isocratic gels (see Note 5). PAGE
provides a simple but powerful approach for separating the saccharide products generated
in the sequencing process.
3.4.1. Preparing the PAGE Gel
1. Assemble the gel unit (consisting of glass plates and spacers, etc).
2. Prepare and degas the resolving gel acrylamide solution without ammonium persulfate or
TEMED. To make a 30% acrylamide gel solution for a 16 cm × 12 cm × 0.75 mm gel,