Quantification of Uric Acid or Xanthine in Plant Samples

We developed this protocol to assay and quantify the content of uric acid or xanthine in various tissues of Arabidopsis thaliana mutant lines with defective urate oxidase or xanthine dehydrogenase1 and in their complementation and suppressor lines (Hauck et al., 2014). The protocol is based on a met...

Full description

Saved in:
Bibliographic Details
Published in:Bio-protocol 2015-07, Vol.5 (13)
Main Authors: Hauck, Oliver, Witte, Claus-Peter
Format: Article
Language:English
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:We developed this protocol to assay and quantify the content of uric acid or xanthine in various tissues of Arabidopsis thaliana mutant lines with defective urate oxidase or xanthine dehydrogenase1 and in their complementation and suppressor lines (Hauck et al., 2014). The protocol is based on a method developed by Invitrogen Life Technologies for measuring uric acid or xanthine in human serum (see References 2 and 3). That protocol though required two adaptions for its use in plant science. Firstly by heating the plant samples, the activity of urate oxidase and xanthine dehydrogenase in the wild type samples is eliminated. Wild type extracts always serve as the proper pigmentation background when calculating the standard curves of uric acid and xanthine. Secondly, all samples are measured with and without the addition of urate oxidase or xanthine dehydrogenase to correct for any H2O2 in the samples induced by previous stress.The assay is based on the following pair of coupled reactions:1) Uric acid + O2 → Hydroxyisourate + H2O2 (urate oxidase reaction)2) AR + H2O2 → Resorufin + O2 (horse radish peroxidase reaction)Accordingly for Xanthine:1) Xanthine + H2O + O2 → Uric acid + H2O2 (xanthine oxidase reaction)2) AR + H2O2 → Resorufin + O2 (horse radish peroxidase reaction)
ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.1523