A quantitative colorimetric assay for semialdehydes.
We have studied the ability of the enzyme pyrroline-5-carboxylate dehydrogenase to oxidize aldehydes which are structurally similar to pyrroline-5-carboxylate, the enamine of gamma-glutamyl semialdehyde. These studies required the organic synthesis of semialdehydes not commercially available. Accurate quantitation of the concentration of these compounds in solution is necessary because they polymerize upon evaporation to dryness. Reported here is a method for colorimetric measurement of semialdehydes in solution by formation of an oxidized covalent adduct with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole. This adduct after oxidation produces a product that has an absorption maximum at 302 +/- 2 nm. This characteristic absorbance maximum was not found in the spectra of the covalent adducts of simple aliphatic aldehydes with the chromophore. Using glyoxylic acid as standard, the values obtained for the concentration of semialdehydes in solution were independently verified from quantitation of the proton concentration of the oxidized adducts by integration of the area under the peaks of their 1H NMR signals. These latter values were compared to the signal strengths of an internal standard. The values obtained by the colorimetric and NMR techniques were in excellent agreement, thus allowing use of the colorimetric technique as a simple method for routine laboratory analysis of semialdehydes.
Small
W C
WC
Department of Biochemistry and Nutrition, School of Medicine, University of North Carolina, Chapel Hill 27599-7260.
Jones
M E
ME
D000447:Aldehydes / Q000032:analysis* / Q000138:chemical synthesis; D003124:Colorimetry / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds
D000447:Aldehydes; D013438:Sulfhydryl Compounds; C006537:4-amino-5-hydrazino-1,2,4-triazole-3-thiol