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 oligoarabinoside 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(OH)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.
Campargue
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Centre de Biologie et Physiologie Végétales, UMR CNRS, UPS 5546, Université Paul Sabatier, Toulouse, France.
Lafitte
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Esquerré-Tugayé
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Mazau
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D000837:Anion Exchange Resins; D001089:Arabinose / Q000032:analysis*; D002473:Cell Wall / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004563:Electrochemistry; D006909:Hydroxyproline / Q000032:analysis*; D010944:Plants / Q000737:chemistry*
D000837:Anion Exchange Resins; D001089:Arabinose; D006909:Hydroxyproline