Yield:-

Reaction Conditions:

with Azotobacter vinelandii globin-coupled sensor O₂-ligated ferrous form;sodium chloride;magnesium chloride in aq. buffer at 37; pH=7.5;Kinetics;Enzymatic reaction;Concentration;

Steps:

DGC assay

A HPLC-based DGC assay was adapted from Ref. (54) and performed in triplicate to quantify c-di-GMP synthesis by AvGReg. Shortly, 10 μM of enzyme was incubated at 37°C with 20-65 μM GTP in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 10 mM MgCl2. Aliquots were taken at different times, and the reaction was rapidly stopped with one-fourth volume of 0.5 M EDTA, pH 8.0, which ensures repeatability in the measurement of more time points in a few seconds time frame, as required for initial rate determination of enzymatic processes. For deproteination, the stopped reaction mixture was heated for 5 min at 95°C and centrifuged for 10 min at 11,300 g. Twenty microliters of the supernatant was injected onto an Adsorbosphere Nucleotide-Nucleoside RP C18 HPLC column (250 · 4.6 mm; Alltech) equipped with the same type guard column (7.5 · 4.6 mm; Alltech/Grade). As mobile phase, 0.15 M NaH2PO4 and 40% acetonitrile was used. A relatively rapid linear gradient from 0% to 35% acetonitrile, for 10 min at 1 mL*min-1, was applied to separate GTP, c-diGMP, and pGpG, with which we could efficiently process the high number of samples produced. Known amounts of all three molecules were used for standardization.

References:

Germani, Francesca;Nardini, Marco;De Schutter, Amy;Cuypers, Bert;Berghmans, Herald;Van Hauwaert, Marie-Louise;Bruno, Stefano;Mozzarelli, Andrea;Moens, Luc;Van Doorslaer, Sabine;Bolognesi, Martino;Pesce, Alessandra;Dewilde, Sylvia [Antioxidants and Redox Signaling,2020,vol. 32,# 6,p. 378 - 395]