Small W C, Jones M E
Department of Biochemistry and Nutrition, School of Medicine, University of North Carolina, Chapel Hill 27599-7260.
Anal Biochem. 1990 Feb 15;185(1):156-9. doi: 10.1016/0003-2697(90)90272-b.
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.
我们研究了脯氨酸-5-羧酸脱氢酶氧化与脯氨酸-5-羧酸结构相似的醛类的能力，脯氨酸-5-羧酸是γ-谷氨酰半醛的烯胺。这些研究需要有机合成市售可得的半醛。准确测定溶液中这些化合物的浓度是必要的，因为它们在蒸发至干时会聚合。本文报道了一种通过与4-氨基-3-肼基-5-巯基-1,2,4-三唑形成氧化共价加合物来比色测定溶液中半醛的方法。该加合物氧化后产生一种在302±2nm处有最大吸收的产物。在简单脂肪醛与发色团的共价加合物光谱中未发现这种特征性的最大吸收。以乙醛酸为标准品，通过对氧化加合物1H NMR信号峰下面积积分来定量质子浓度，从而独立验证了溶液中半醛浓度的测定值。将这些后者的值与内标物的信号强度进行比较。通过比色法和核磁共振技术获得的值非常一致，因此可以将比色法作为一种用于半醛常规实验室分析的简单方法。
