Qs quartz cuvette

The activity measurements were performed using a Varian Cary Eclipse Fluorescence Spectrophotometer. For activity measurements, the hTPH2 variants were thawed under running water, filtered, and the concentrations were determined by UV‐Vis absorption at 280 nm. The hTPH2 samples were diluted to a protein concentration of 5 μm in the buffer in which it was purified. TPH2 activity was assayed in a reaction mixture (10 × 10 mm QS quartz cuvette from Hellma (Müllheim, Germany) – 2500 μL total volume) containing 50 mm HEPES/NH₄OH, 200 mm (NH₄)₂SO₄, pH 7.0, 0.025 g·L⁻¹ catalase, 25 μm (NH₄)₂Fe(II)(SO₄)₂^.6H₂O, 7 mm dithiothreitol (DTT), 60 μm l‐Trp, and 300 μm BH₄ with stirring at 15 °C 14, 51. The excitation wavelength was 300 nm, and the emission was monitored at 330 nm. The activities were determined by the initial slope (intensity·min⁻¹) of the monitored fluorescence and were quantified using varian spectrophotometer software (Agilent Technologies). Quantification occurred through linear regression on a manually placed interval of minimum 0.04 min of the initial curve. Denaturation was performed by heating the protein in aliquots of 1300 μL to 30 °C in a water bath. The denaturation was stopped by cooling the protein solution in ice water.

The wt and mutant TEM variants were generated as outlined in the ESI.† The proteins were expressed in E. coli using a bespoke plasmid based on pBAD called pBADKAN, and purified as described in the ESI.† The TEM-dependent kinetics of ampicillin hydrolysis (ε₂₃₅ = 1500 M^–1 cm^–1) were determined spectrophotometrically using 1 cm path length QS quartz cuvette (Hellma). Hydrolysis assays were carried out in a 1 mL reaction volume. Purified enzyme was diluted to a final concentration of 250 ng μL^–1 in 50 mM sodium phosphate buffer, pH 8 at room temperature. Reactions were started by addition of ampicillin and hydrolysis was measured by the decrease in absorbance at 235 nm. Ampicillin concentrations ranged from 50 μM to 800 μM. Kinetic parameters were calculated using initial rate of hydrolysis at each substrate concentration and then fitting to the Michaelis–Menten equation using GraphPad Prism. TEM β-lactamase activity using the colorimetric substrate nitrocefin was performed essentially as described previously.^{47 (link),48 (link)}

Far-UV CD spectra were recorded on a JASCO J-810 spectropolarimeter at 20 °C, using 50 µM of the Tsr2 variants in NMR buffer in 1 mm QS quartz cuvettes (Hellma Analytics) and scanning from 260 to 190 nm. The results were displayed as molar ellipticity [θ] with units of [deg cm² dmol^–1]. The thermal denaturation curves were obtained by following the molar ellipticity at 222 nm while increasing the temperature by 1 °C per minute from 20 to 93 °C.