A Modified Kulka Micromethod for the Rapid and Safe Analysis of Fructose and 1-Deoxy-d-xylulose-5-phosphate

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.

New Approach for the Construction and Calibration of Gas-Tight Setups for Biohydrogen Production at the Small Laboratory Scale

“…To remove proteins from the medium supernatant, 250 µl medium supernatant were precipitated with 17 µL of a 72% (w/v) trichloroacetic acid solution (TCA: final concentration: 4.6% (w/v)), incubated for 30 min at room temperature, and the protein pellet removed by a 20 min centrifugation step (11,000× g). The fructose content in 10 µL to 20 µL aliquots of the deproteinized supernatant was determined by performing the modified Kulka method as described in detail by Shaw and Ghosh [34].…”