Separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length by anion-exchange high-performance liquid chromatography.
A method for separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length is described. Oligomers were labeled at the reducing end with 2-aminopyridine and then analyzed by anion-exchange high-performance liquid chromatography using a sodium acetate gradient. The amount of each oligogalacturonide present was determined by comparison to the response of an internal reference oligogalacturonide over a range from 0.5 to 20 nmol per oligomer. At least 5 h of incubation in the 2-aminopyridine reagent was required to obtain maximum and oligomer length-independent derivatization. To be analyzed using this technique, oligogalacturonides must possess a reducing terminus, they should be deesterified prior to derivatization if identification of the actual galacturonide chain length is desired, and they should fall within the range of 3 to over 25 galacturonide residues per oligomer. The wide range of oligogalacturonides separable, sensitivity of detection, ease of quantitation of chromatographic data, and ability to hydrolyze the 2-aminopyridinyl group from sugars makes this technique of potential use for numerous applications ranging from simple characterization of oligogalacturonide mixtures to purification of oligomers for use in bioassays.
Maness
N O
NO
Department of Biochemistry, Oklahoma State University, Stillwater 74078.
Mort
A J
AJ
D002852:Chromatography, Ion Exchange; D006603:Hexuronic Acids / Q000032:analysis / Q000302:isolation & purification*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D010368:Pectins; D014574:Uronic Acids / Q000302:isolation & purification*
D006603:Hexuronic Acids; D014574:Uronic Acids; C007819:galacturonic acid; D010368:Pectins; C003181:polygalacturonic acid