Abstract
The Kulka resorcinol assay (Kulka, R.G., Biochemistry 1956, 63, 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.
Highlights
The determination of sugars in biological assays is a common task in many areas of biochemistry, fermentation, and more recently, systems biology
We have not examined these effects systematically, since our results indicate that fructose measurements in the presence of about 100-fold higher concentrations of glucose will be unreliable and should be determined by another method
We have shown previously [22,23] that the purple bacterium Rhodospirillum rubrum will grow semi-aerobically in the dark in shake flasks in the presence of the carbon sources succinate and fructose
Summary
The determination of sugars in biological assays is a common task in many areas of biochemistry, fermentation, and more recently, systems biology. For studies requiring the rapid analysis of a large number of samples, e.g., in areas of enzyme kinetics, fermentation, or systems biology, colorimetric methods involving usually absorbance or fluorescence measurements are still of great utility due to their potential for high-throughput as well as being suitable for automated techniques. Many methods for the colorimetric measurements of furanoses and pyranoses exist, some of which involve direct measurement of extracted samples [8,9,10,11,12,13,14,15], whereas others require prior rapid purification by thin-layer chromatography [16,17,18,19] Many of these methods are today, in view of laboratory safety regulations, only of historical importance, as they usually involve large quantities of acids or reagents with significant potential as carcinogens
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