2-4-dinitrophenylhydrazine and phenylhydrazine

Allantoinase hydrolyzes allantoin, a purine metabolite and a nitrogen transport 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 phenylhydrazone to 1, 5-diphenylformazan that is measured colorimetrically. When used with crude cell extracts this assay is problematic and its complexity is a hindrance to detailed enzyme characterization; thus, three alternative assays were developed. In the first assay, 2, 4-dinitrophenylhydrazine was reacted with allantoate-derived glyoxylate and the concentration of hydrazone was measured directly by its absorbance at 450 nm. This assay exhibited enhanced reproducibility compared to the standard method and entailed fewer steps, but was 3-fold less sensitive. The second assay combined allantoate 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-fold less sensitive than the standard assay. The third assay involved urease-catalyzed hydrolysis of allantoate-derived urea, followed by reaction of the released ammonia to form indophenol. This was the most laborious of the assays, but was more sensitive than the standard method.

Topics: Amidohydrolases; Phenylenediamines; Phenylhydrazines; Substrate Specificity; Urease

Soybean lipoxygenase-1 was reinvestigated with respect to its quinoprotein nature. It has been reported previously that soybean lipoxygenase-1 contains pyrroloquinoline quinone as the organic cofactor. Because spectroscopic data were found to be inconsistent with the evidence presented in [1], we sought to reproduce the published data by carefully following the procedures described in [1] and supplementing them with new analytical results. The combined data lead us to conclude that soybean lipoxygenase-1 is not a quinoprotein.

Topics: Coenzymes; Formazans; Glycine max; Hexanols; Lipoxygenase; Phenylhydrazines; PQQ Cofactor; Quinolones; Spectrophotometry

Topics: Aldehydes; Chemical Phenomena; Chemistry; Keto Acids; NAD; NADP; Nucleotides; Phenylhydrazines; Pyridines; Research; Ribose; Spectrum Analysis

Topics: Ascorbic Acid; Hydrazines; Phenylhydrazines; Temperature

Topics: Adrenal Glands; Androgens; Animals; Castration; Gonadal Steroid Hormones; Humans; Hydrazines; Male; Phenylhydrazines; Rats; Rosaniline Dyes; Staining and Labeling; Testis