Superdex 200 increase 5 150 gl

The SEC-MALS system was comprised of HPLC system coupled with UV and fluorescence detector (Shimadzu), Dawn Heleos II Multi-Angle Light Scattering detector (Wyatt) and Optilab T-rEX refractive index (RI) detector (Wyatt). Superdex 200 increase 5/150 GL (GE) was used to separate the species in solution. The system was pre-equilibrated with buffer containing 20 mM HEPES (pH 7.5), 500 mM NaCl and the RI detector was purged for 2 column volumes until the dRI value was stable. The dRI value was used for data analysis later. Protein samples were dissolved in 50 μL of the same buffer with at least 2 mg/mL concentration and filtered through 0.1-μm filter before injection. Astra software was used for sample auto-injection and data analysis.

¹⁵N‐labeled MOZART1 protein buffered in 50 mM sodium phosphate pH 6.0, 150 mM NaCl was analyzed on a Superdex 200 Increase 5/150 GL (GE Healthcare) column with multiangle light scattering (MALS). The column was equilibrated in a 50 mM sodium phosphate 0.1 μm filtered buffer (pH 6.0 and 150 mM NaCl) on an Agilent 1260 Infinity LC chromatographic system (Agilent Technology). Data were collected using a DAWN 8+ and Optilab T‐rEX refractive index detector (Wyatt Technology). Of 150 µM protein sample, 70 µL was loaded on the column and the separation was performed at a flow rate of 0.4 mL/min at 15°C. Results were analyzed using the ASTRA 6.1 software (Wyatt Technology).

The respective proteins were mixed in equimolar quantities and incubated for 10 min at 4 °C in 100 mM sodium citrate pH 5.75 and in the presence or absence of the respective effector molecules phenylalanine (15 mM) or BH4 (0.2 mM). Ten to forty micrograms of protein was injected into an analytical SEC column, Superdex 200 Increase 5/150 GL (GE Healthcare), using an ÄKTAmicro system. The system was run at 0.2 mL/min, and the mobile phase consisted of 100 mM sodium citrate pH 5.75 and respective effector molecules. The elution of hGCH1 or hGCH1−hGFRP complexes was detected via absorbance measurements at 280 nm using an ultraviolet absorbance detector. Comparative analysis of retention times of wild-type GCH1−GFRP in the absence and presence of effector molecules as well as single-component injections were used to determine successful complex formation of mutated GCH1.