Shaw Shreya, Ghosh Robin
Department of Bioenergetics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany.
Metabolites. 2018 Nov 8;8(4):77. doi: 10.3390/metabo8040077.
The Kulka resorcinol assay (Kulka, R.G., 1956, , 542⁻548) for ketoses has been widely used in the literature but suffers from two major disadvantages: (a) it employs large amounts of potentially harmful reagents for a general biology laboratory environment; and (b) in its original formulation, it is unsuited for modern high-throughput applications. Here, we have developed a modified Kulka assay, which contains a safer formulation, employing approx. 5.4 M HCl in 250 µL aliquots, and is suitable for use in high-throughput systems biology or enzymatic applications. The modified assay has been tested extensively for the measurement of two ketoses-fructose (a common substrate in cell growth experiments) and 1-deoxy-d-xylulose-5-phosphate (DXP), the product of the DXP-synthase reaction-which until now has only been assayable using time-consuming chromatographic methods or radioactivity. The Kulka microassay has a sensitivity of 0⁻250 nmol fructose or 0⁻500 nmol DXP. The assay is suitable for monitoring the consumption of fructose in bacterial growth experiments but is too insensitive to be used directly for the measurement of DXP in in vitro enzyme assays. However, we show that after concentration of the DXP-enzyme mix by butanol extraction, the Kulka resorcinol method can be used for enzyme assays.
用于检测酮糖的库尔卡间苯二酚测定法（库尔卡，R.G.，1956年，第542 - 548页）在文献中已被广泛使用，但存在两个主要缺点：（a）对于一般生物学实验室环境而言，它使用了大量具有潜在危害的试剂；（b）其原始配方不适用于现代高通量应用。在此，我们开发了一种改良的库尔卡测定法，它采用更安全的配方，在250微升等分试样中使用约5.4 M盐酸，适用于高通量系统生物学或酶学应用。该改良测定法已针对两种酮糖——果糖（细胞生长实验中的常见底物）和1 - 脱氧 - D - 木酮糖 - 5 - 磷酸（DXP，DXP合酶反应的产物）的测量进行了广泛测试，而DXP此前只能使用耗时的色谱方法或放射性检测方法进行测定。库尔卡微量测定法对果糖的检测灵敏度为0 - 250纳摩尔，对DXP的检测灵敏度为0 - 500纳摩尔。该测定法适用于监测细菌生长实验中果糖的消耗情况，但灵敏度太低，无法直接用于体外酶学测定中DXP的测量。然而，我们表明，通过丁醇萃取浓缩DXP - 酶混合物后，库尔卡间苯二酚法可用于酶学测定。
