Specord200 uv vis spectrophotometer

Absorbance‐based measurements were performed with a Specord200 UV/Vis spectrophotometer (Analytik Jena) as described previously (Shydlovskyi et al, 2017). Proteins were diluted in buffer C supplemented with 50 μM BSA and incubated for 5 min in a temperature‐controlled cuvette at 25°C. Nucleotides were injected into the cuvette at final concentrations of 1 mM (GTP, GppNHp, GTPγS) or 250 μM (GDP·AlF_X). Polymerization of hGBP1 was followed as absorbance signal at 350 nm over time. In experiments with GTP, the nucleotide composition of the sample was analyzed at defined time points. To do so, 5 μl aliquots were taken from the cuvette and GTPase hydrolysis was immediately stopped by addition of 10 μl of 10% H₃PO₄, followed by neutralization with 30 μl of 0.77 M K₂HPO₄. Nucleotide composition was analyzed via separation of GTP, GDP, and GMP by reversed‐phase high‐performance liquid chromatography (HPLC) using a Chromolith Performance RP‐18 endcapped column (Merck) connected to a BT4100 HPLC‐pump (Shimadzu). Retention times of nucleotides were detected via monitoring the absorption at 254 nm with a MD‐2010 Plus multi wavelength detector (Jasco). To quantify the concentration of GMP, GDP, and GTP, peak areas corresponding to the respective nucleotide were integrated with the ChromPass software (Jasco).

The thickness and refractive index of the layers (on glass) were calculated by fitting a thin-layer optical model the transmittance spectra (Hild method [28 (link)]). The spectra were recorded with 10 nm/s measuring speed, and 1 nm resolution from 350 to 1100 nm by an Analytik Jena Specord 200 UV–Vis spectrophotometer. The thickness and refractive index were calculated from the fitted values of twelve replicate samples (n = 12).

The TBS was used to measure the in vitro release of Ca, P, and EGCG from E/PA@HMS to eliminate the interference from Ca and P existing in artificial saliva, simulated body fluid, or PBS. Specifically, E/PA@HMS (100 mg) was immersed in 10 mL of TBS at pH 7.4. The amounts of Ca and P released were detected at 0.5, 1, 3, 5, 7, 14, 21, and 28 days, respectively. This mixture at each time interval was centrifuged at 4000 rpm for 5 min. One mL aliquot of supernatant was immediately collected and supplemented by fresh TBS at an equal volume [37 ]. These collected aliquots were analyzed by a Specord200 UV–vis spectrophotometer (Analytik Jena, Germany) at a 325 nm wavelength to determine the content of released EGCG. Meanwhile, the aliquots were analyzed by Intrepid II XSP inductively coupled plasma-atomic emission spectroscopy (ICP-AES, Thermo Fisher, USA) to measure the contents of released Ca and P.