Neslab rte 7 circulator

Stopped-flow fluorescence measurements were performed using RX.2000 Rapid Mix Accessory (Applied Photophysics Ltd., Leatherhead, UK) with a dead time of 6 ms. Temperature of all solutions was kept at 20 °C using NESLAB RTE-7 circulator (Thermo Scientific) and Peltier-controlled cell holder. Equal volumes (0.3 mL) of 5–18 µM rWT/C18S Ocm (Ca²⁺ to protein molar ratio of 2:1, or 5:1 for Mg²⁺ and the metal-depleted protein) and 1–2 mM EGTA/EDTA in 10 mM HEPES-KOH buffer, pH 8.2 (Ca²⁺ dissociation experiments) or pH 7.3 (Mg²⁺ dissociation), were mixed using a pneumatic drive. To prevent Ca²⁺ contamination of Ocm solution during the Mg²⁺ dissociation studies, 1 mM EGTA was added. Tyr fluorescence emission was measured at 305 nm. Each experiment was repeated 4 times. The experimental data were fitted within the sequential metal binding scheme [IV] [2 ] as described earlier [53 ].

Estimation of pK_a value of Cys18 of Ocm at 20 °C was carried out spectrophotometrically mainly as described in ref. [39 (link)]. UV absorption spectra of rWT/C18S Ocm (23 μM) were measured with a Cary 100 spectrophotometer (Varian Inc., Palo Alto, CA, USA), equipped with NESLAB RTE-7 circulator (Thermo Scientific, Waltham, MA, USA). Buffer conditions: 30 mM MES, 30 mM HEPES, 30 mM H₃BO₃, 30 mM glycine, 100 mM KCl, 1.5 mM EDTA (apo-Ocm), or 1 mM CaCl₂ (Ca²⁺-bound Ocm). The protein solution was titrated by small aliquots of KOH stock solution. To avoid solution contamination with Ca²⁺ during the titrations, pH values were independently measured under identical conditions. The protein absorbance at 240 nm was corrected for a minor light scattering contribution via extrapolation with power function of the long wavelength region of Ocm absorption spectrum. Contribution of deprotonated Tyr residues to the absorbance at alkaline pH values [40 (link)] was subtracted from the experimental data.