Protein Protocols Protein Protocols

Sequencing Heparan Sulfate Saccharides 901
Fig. 4. HPLC purification and IGS of a HS decasaccharide. (A) SAX-HPLC of a pool of HS
decasaccharides derived by heparitinase treatment of porcine mucosal HS (for details see
ref. 11). The arrowed peak was selected for sequencing. (B) IGS of the purified HS
decasaccharide on a 16-cm 33% gel using the combinations of pHNO 2 and exoenzyme treatments
indicated (track 1, untreated, 20 pmol; other tracks correspond to approx 400 pmol per
track of starting sample for pHNO 2 digest). (C) Determining the sequence of the nonreducing
disaccharide unit of the HS decasaccharide using the mercuric acetate (MA) and G6Sase treatments
shown (approx 40 pmol per track; track 1, untreated). (From ref. 11, Copyright 1999
National Academy of Sciences, USA.)
the nitrous acid treatment. They are probably due to loss of an N-sulfate group, and
normally these do not affect interpretation of the shifts in the major bands (11).
If the saccharide being sequenced was derived by bacterial lyase treatment, it will
have a ∆4,5-unsaturated uronate residue at its nonreducing terminus. If this residue has
a 2-O-sulfate attached, this can be detected by susceptibility to I2Sase (see Fig. 3B),
but the sugar residue itself is resistant to both Idase and Gase. Its removal is required to
confirm whether there is a 6-O-sulfate on the adjacent nonreducing end glucosamine
(see Figs. 3B and 4C for examples). Bacterial enzymes that specifically remove the
∆4,5-unsaturated uronate residues (and the 2-O-sulfate groups that may be present on
them) are now available commercially (see Table 1). Alternatively, they can be removed
chemically with mercuric acetate (21; see Figs. 3B and 4C).
In addition to the basic sequencing experiment, it is wise to confirm agreement of
the data with an independent analysis of the disaccharide composition of the saccharide
(11). It can sometimes be difficult to sequence the reducing terminal monosaccharide
owing to it being a poor substrate for the exoenzymes. In these cases it has proved
more effective to analyze the terminal 2AA-labeled disaccharide unit in comparison to
2AA-labeled disaccharide standards (11).