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 called extensin, contains arabinose and oligoarabinaside side chains O-glycosidically linked to hydroxyproline (Hyp). We present a highly sensitive method for determining both the glycosylation pattern and the Hyp content of HRGP requiring only nanomole amounts of each Hyp-compound for accurate determination. This method is based on anion-exchange chromatography followed by pulsed amperometric detection of the Hyp-oligoarabinosides 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 isolation of HRGP. (C) 1998 Academic Press.