Colorimetric Determination of Tungsten and Molybdenum in Biological Samples.

Abstract

Molybdenum (Mo) and tungsten (W) are elements that are utilized in biological systems. They are typically incorporated into the catalytic sites of enzymes coordinated to an organic pyranopterin cofactor; Mo may also be present in the form of a FeMo cofactor. While Mo is used by all branches of life, only a few microbes are able to utilize W. In order to study Mo- and W-dependent enzymes, it is important to be able to measure Mo and W in biological samples. Methods for determining Mo and W content in biological samples currently involve expensive and time-consuming processes like inductively coupled plasma mass spectrometry (ICP-MS) and chelation ion chromatography. There are less intensive colorimetric methods for measuring W in abiotic samples, but these have not been adapted to biological samples like cytosolic extracts and purified proteins. Herein, we developed a colorimetric assay based on the complexation of quercetin to molybdate (MoO₄ ^2-) or tungstate (WO₄ ^2-), the oxyanion forms of Mo and W that readily form in denatured biological samples. In the assay, the absorbance of quercetin is redshifted proportionally to the concentration of tungsten or molybdenum, which can be measured spectrophotometrically. This protocol provides a rapid method for screening biological samples for both Mo and W, although it does not distinguish between them. Key features • This protocol is adapted from the method developed by El-Sayed et al. [1] for analyzing abiotic samples. • The protocol is designed for a 96-well plate format and optimized for analyzing protein samples. • Can be used over the range of 1-20 μM W or Mo. • Allows for rapid and high throughput Mo and W determination in samples during protein purification. Graphical overview Overview of colorimetric determination of tungsten in protein samples using quercetin. Protein samples (200 μL) are transferred to microcentrifuge tubes along with 50 μL of 2.5× nitric acid mix and incubated at 65 °C overnight. The tubes are spun at 20,000× g for 20 min and 200 μL of the supernatant is transferred to a holding plate. 40 μL of each sample is transferred from the holding plate to four wells on an optical plate using a multichannel pipette. 160 μL of ethanol blank mix is added to one well per sample and 160 μL of quercetin mix is added to the remaining wells of each sample. The plate is sealed and shaken to mix each well. The seal is removed, and absorbances at 419 nm of each well are measured in a plate reader.