Journal: FEBS Open Bio

Article Title: Characterization of an intertidal zone metagenome oligoribonuclease and the role of the intermolecular disulfide bond for homodimer formation and nuclease activity

doi: 10.1002/2211-5463.12720

Figure Lengend Snippet: Effect of Mn 2+ (A) and Mg 2+ (B), pH (C), and NaCl (D) on the nuclease activity of MG Orn. The effect of the metal ions and pH on the enzyme activity has been determined using the time‐resolved p NP ‐ TMP activity assay at 25 °C as described in with 1.1 μg Orn and varying amounts of Mn 2+ and Mg 2+ as well as 1.5 μg MG Orn and pH values from 6.5 to 9 using MES ( pH 6.5), HEPES ( pH 7–7.5), and Tris ( pH 8–9) as indicated in A–C. The rate of hydrolysis of p NP ‐ TMP at the varying pH values was calculated according to Hamdan et al . . Error bars indicate the standard deviation of the measurements. The effect of NaCl in a range of 0–2 m is shown in (D) and has been tested at 25 °C with the gel‐based nuclease activity assay with 0.05 μ m 7mer RNA substrate and 0.8 μg MG Orn in 50 m m Tris‐ HC l pH 8.0, 0.2 mg·mL −1 BSA , 2% glycerol and 1 m m MnCl 2 for 15 min. Samples were analyzed on 20% denaturing PAA gels (8 × 8 cm). Reaction buffer was used as negative control (Neg) instead of protein solution.

Article Snippet: The reactions contained 50 m m HEPES pH 7.5 (at 25 °C), 72 m m NaCl, SYPRO ® Orange (Merck KGaA) in a final dilution of 6× and 4 μg of protein.

Techniques: Activity Assay, Standard Deviation, Negative Control