UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module

Abstract

Despite the prevalence of ortho- and pyrophosphate in biochemistry, operationally simple and versatile high-throughput methodologies for their quantification are lacking. We herein introduce PUB, a module for p hosphate detection by continuous U V-spectroscopic monitoring of 5- b romouridine phosphorolysis. The PUB module uses cheaply available, bench-stable reagents and can be employed for continuous and discontinuous reaction monitoring in biochemical assays to detect (pyro-)­phosphate concentrations spanning almost 4 orders of magnitude, as demonstrated with representative use cases.

Quality Data from Messy Spectra: How Isometric Points Increase Information Content in Highly Overlapping Spectra

“…The data in a belong to 5‐nitrouracil and its monoanion (previously unpublished data) and serve as an example of an arbitrary transformation [8] . The data in b were taken from our recent publication about phosphate detection with the PUB module [9,10] . The data in the remaining panels were originally published by Hutchinson, Welsh and Burés ( c ), [11,12] Lei and colleagues ( d ), [13,14] and Bramham, Zalar, and Golovanov ( e ), [15,16] and I am grateful to the authors for generously providing the raw data upon request.…”

“…Isosbestic points are frequent because they only require the absorption spectra of the starting material and the product to intercept. For instance, the UV absorption spectra of the nucleobase 1 b and its corresponding monoanion 1 b − (acquired at identical concentrations) intercept at 286 nm [9] . Thus, absorption spectra of 1 b at different pH values (essentially following the deprotonation of 1 b ) will exhibit a constant extinction at 286 nm while the remainder of the spectrum will reflect that of 1 b at lower pH values and that of 1 b − at higher pH values (Figure 1b).…”

Alternative Assay Reagents for UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module

“…The general procedures concerning solutions, UV analyses, equations, and enzymatic reactions are available in detail from the Supporting Information of PUB …”

“…The temperature-dependence of the extinction coefficients was determined with 50 and 100 μM solutions of nucleoside and nucleobase in 200 mM glycine buffer pH 9 (200 μL total volume) in microcuvettes, in analogy to the procedure described previously …”

“…Motivated by the lack of cheaply available, operationally simple, and versatile high-throughput methods for continuous phosphate detection in biochemical assays, we recently introduced a module for phosphate detection via UV-spectroscopic monitoring of 5-bromouridine phosphorolysis, termed PUB . The PUB module uses commercially available 5-bromouridine and is capable of detecting inorganic (pyro‑)­phosphate over a concentration range spanning nearly 4 orders of magnitude, while being readily compatible with parallelized experimentation.…”