Cary 100 bio uv vis spectrometer

Unless otherwise stated, steady-state kinetic assays were carried out at 25 °C in a 1.5 mL tube with 30 μg of Cu(OH)₂ SCs (3.5 × 10⁹ supercages) or 300 ng HRP (4.1 × 10¹² enzyme molecules) in 500 μL of reaction buffer (0.2 M NaAc, pH 4.5) in the presence of 530 μM H₂O₂ for Cu(OH)₂ SCs or HPR using 800 μM TMB as the substrate. For experiments at different pHs (1–12) at 25 °C, 30 μL of H₂O₂ (30%) was added to 400 μL of reaction buffer and vortexed for 4 min. Then, 40 μL of TMB (10 mM) was added into the mixture and vortexed for another 4 min. Finally, 30 μL of Cu(OH)₂ SCs (1 mg/mL) was quickly added to the mixture. Immediately after addition of Cu(OH)₂ SCs, color changes were observed. All reactions were monitored in time-scan mode at 652 nm using a Cary Bio-100 UV/vis spectrometer (Varian).
To study the effect of different temperatures (22–65 °C) at pH 4.5, 400 μL of reaction buffer was held at the desired temperature for 5 min. Then, 30 μL of H₂O₂ (30%) was added to the reaction buffer and vortexed for 1 min, and the mixture was held at that temperature for 4 min. Then, 40 μL of TMB (10 mM) was added to the mixture and vortexed for 1 min, and the mixture was held at that temperature for another 4 min. Finally, 30 μL of Cu(OH)₂ SCs (1 mg/mL) was quickly added to the mixture. The reaction was monitored in time-scan mode at 652 nm using a Cary Bio-100 UV/vis spectrometer (Varian).

Absorption spectra of all protein samples were obtained at 20 °C using a Cary 100 Bio UV-Vis spectrometer. The concentration of the protein samples was kept at around 80 μM in either H₂O or D₂O buffer (50 mM NaH₂PO₄, 10 mM NaCl, pH/D 8.0). Dark-adapted spectra were obtained first and then the samples were illuminated with ~500 mW of 455 (± 10) nm light for ~ 1 min in order to generate the light state.

All activity assays and steady-state kinetics were performed on a Cary 100 Bio UV-Vis spectrometer equipped with a temperature controller and magnetic stirring. Transient-state kinetics was carried out on an Applied Photophysics SX20 stopped-flow spectrometer equipped with sequential mixing, a PDA detector, and a monochromator. All chemical and biochemical reagents were purchased at the highest grade and used without further purification. Protein concentrations were determined by bicinchoninic acid assays (BCA).^{29 (link)} Stocks of H₂O₂ and ascorbate were prepared fresh before experiments. Concentrations of H₂O₂ were determined at 240 nm using ε₂₄₀ = 43.6 M⁻¹cm⁻¹. Buffers of pH 2.0–6.5, 7.0–8.0, 8.5–9.0, and 9.5–11.5 were prepared using sodium citrate, potassium phosphate, Tris-HCl, and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), respectively. All kinetic measurements were performed in triplicate and data were processed by OriginPro 2015.