ANALYSIS OF HYDROXYPROLINE AND HYDROXYPROLINE-ARABINOSIDES OF PLANT-ORIGIN BY HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY PULSED AMPEROMETRIC DETECTION

The plant cell wall hydroxyproline-rich glycoprotein (HRGP), also call ed extensin, contains arabinose and oligoarabinaside side chains O-gly cosidically linked to hydroxyproline (Hyp). We present a highly sensit ive method for determining both the glycosylation pattern and the Hyp content of HRGP requiring only nanomole amounts of each Hyp-compound f or accurate determination. This method is based on anion-exchange chro matography followed by pulsed amperometric detection of the Hyp-oligoa rabinosides and Hyp released from HRGP by 0.22 M Ba(ON)(2) hydrolysis, which cleaves only peptidyl bonds. A sodium acetate gradient. (0-250 mM) in 150 mM NaOH elutes Hyp and the Hyp-oligoarabinosides Hyp-(Ara)( 1-5) in less than 40 min. We have used this procedure to determine the glycosylation pattern of Hyp in plant cell walls, without prior isola tion of HRGP. (C) 1998 Academic Press.