Figure 1.

Reverse-Phase HPLC Chromatography of APAP1 and Separation of Hyp-O-Glycosides from APAP1 by Size Exclusion Chromatography.

(A) Reverse-phase PRP-1 chromatography profile of AGP-enriched material from Arabidopsis suspension culture medium (modified from Xu et al., 2008). AGPs from the medium of 10-d-old Arabidopsis suspension-cultured cells were purified by sequential DEAE anion exchange and Superose-12 SEC and separated into seven 220 nm-absorbing peaks by reverse-phase PRP-1 chromatography (Xu et al., 2008). The yield of peak 3 (5 mg/5 liters media) was <1% (w/w) of the total AGPs recovered.

(B) The material in peak 3 from (A) was treated with Yariv reagent to yield a Yariv precipitant and a Yariv-soluble fraction. The Yariv-soluble fraction was separated by PRP-1 reverse-phase chromatography into two subfractions, major peak YS1 (1 mg/5 liters media) and shoulder peak YS2 (0.6 to 0.7 mg/5 liters media).

(C) YS1 was hydrolyzed with 0.44 n NaOH at 105°C for 18 h, each hydrolysate was neutralized with 1 n HCl on ice, and freeze-dried residues were dissolved and separated over an analytical Superdex-75 column as described (see Methods). An aliquot (10% volume) of each collected fraction was assayed for Hyp and pentose. Red squares represent absorbance at 560 nm to detect Hyp derivatives; blue diamonds represent absorbance at 665 nm to detect pentose derivatives. The asterisks show the fractions used for more extensive sugar analyses (red asterisks, fractions 22 and 50) and NMR analyses (blue asterisks, fractions 26 and 27).

(D) YS2 hydrolyzed and analyzed as described in (C).