Hyperchromicity assay (HCA)

Specific activities of DNases were determined by HCA according to Kunitz [62]. The 1 ml reaction sample consisted of 50 μg/ml calf thymus DNA dissolved in 10 mM buffer, 0.1 mM CaCl₂ and either 1 mM MgCl₂, CoCl₂ or MnCl₂. The pH-values were adjusted using sodium acetate buffer for pH 5.0–5.5, MES-buffer for pH 6.0–6.5 and Tris-buffer for pH 7.0–9.0. Unless otherwise noted, we routinely used Tris-buffer, pH 7.0, containing 0.1 mM CaCl₂ and 1 mM MnCl₂ for measuring the specific activities of expression or purification samples. The absorbance at 260 nm was recorded for 1 min at RT in a quartz cuvette with 1 cm path length (Beckman DU640). One unit of nuclease is defined by an increase in absorbance of 0.001 per minute under the given conditions. For the calculation of equimolar amounts of the nucleases the molecular mass (MM) of the mature protein was used (Fig 1A). For actin inhibition the respective DNase and monomeric skeletal muscle α-actin were incubated in 50 μl of 10 mM buffer (as indicated in the figure captions) containing 0.1 mM CaCl₂ and 1% (v/v) protease inhibitor cocktail (PIC, Sigma-Aldrich) for 10 min on ice. Afterwards, the sample was completely employed in the HCA.