Assays for allantoinase

Allantoinase hydrolyzes allantoin, a purine metabolite and a nitrogen trans port molecule in plants, to form allantoic acid. The standard enzyme assay involves acid-catalyzed product decomposition to form urea and glyoxylate, reaction of glyoxylate with phenylhydrazine, and oxidative conversion of ph enylhydrazone to 1,5-diphenylformazan that is measured colorimetrically. Wh en used with crude cell extracts this assay is problematic and its complexi ty is a hindrance to detailed enzyme characterization; thus, three alternat ive assays were developed. In the first assay, 2,4-dinitrophenylhydrazine w as reacted with allantoate-derived glyoxylate and the concentration of hydr azone was measured directly by its absorbance at 450 nm. This assay exhibit ed enhanced reproducibility compared to the standard method and entailed fe wer steps, but was 3-fold less sensitive. The second assay combined allanto ate decomposition and glyoxylate reaction with o-phenylenediamine to yield a quinoxalone that was detected by its absorbance at 340 nm. This one-step method was the least error prone of those examined, but was more than 10-fo ld less sensitive than the standard assay. The third assay involved urease- catalyzed hydrolysis of allantoate-derived urea, followed by reaction of th e released ammonia to form indophenol. This was the most laborious of the a ssays, but was more sensitive than the standard method. (C) 1999 Academic P ress.