Abstract
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)
Materials and Reagents
Equipment
Procedure
Representative data
The provided EXCEL-sheet contains the read-out of the plate set-up shown in Figure 2. Use it by pasting your own read-out data into row 39.
Notes
This protocol was developed over the course of several months and found reliable to measure uric acid and xanthine concentrations up to 250 µM in a single assay, because that is the range of a linear relationship between absorption and metabolite concentration (refer to the EXCEL-sheet). When assaying dry seeds, which accumulate the metabolites heavily in the respective enzyme mutants, it may be necessary to make 3- or 5-fold dilutions in a 15-ml tube, before loading the wells.
Recipes
Acknowledgments
This research was funded by the Deutsche Forschungsgemeinschaft (Grant DFG WI3411/1 2) and the German Academic Exchange Service from funds of the German Federal Ministry for Education and Research, program German-Chinese Research Groups.
References
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