Sample managerftn r

Molecular
weights (MWs) were estimated by size exclusion chromatography (SEC)
on an Acquity Arc equipped with a 2998PDA Detector, a Sample Manager
FTN-R, and a Quaternary Solvent Manager-R (Acquity Arc, Waters Corporation,
Milford, MA, USA) with a XBridge TM Protein BEH SEC 200 Å 2.5
μm 4.6 mm × 150 mm column. The standard proteins (Waters
Corporation, Milford, MA, USA) were used to calibrate the column:
uracil (0.112 kDa), ribonuclease A (13.7 kDa), albumin chicken egg
white (44.2 kDa), and thyroglobulin bovine (669 kDa).^{3 (link)} A volume of 10 μL dissolved in 100 mM sodium phosphate
buffer and 0.02% (w/v) sodium azide adjusted at pH 6.8 at a concentration
of 1 mg/mL was injected. Protein elution was recorded by measuring
its absorbance at 280 nm and analyzed with Empower 3 Personal GPC/SEC
software (Waters Corporation, Milford, MA, USA).

The amino acid content was evaluated according to the method of Alaiz et al. with slight modifications [16 (link)]. The samples were hydrolyzed with HCl at 110 °C for 24 h in tubes sealed under nitrogen. Determination of the amino acid content in the acid hydro-lysate was carried out by ultra-high-performance liquid chromatography in a Acquity Arc equipped with a 2998PDA Detector, a Sample Manager FTN-R and a Quaternary Solvent Manager-R (Acquity Arc, Waters corporation, Milford, MA, USA), after derivatization with diethyl ethoxymethylenemalonate, using D,L-α-aminobutiric acid as the internal standard, and a 3 mm × 150 mm reversed-phase column (XSlect^® HSS T3, 2.5 µm) (Waters corporation, Milford, MA, USA). Two solvents: (A) 25-mM sodium acetate and 0.02% sodium azide (pH 6.0) and (B) acetonitrile were used to constitute a binary system gradient. For each amino acid, calibration curves were developed using a mix acid standard at the same hydrolysis conditions of the samples (Merck, Madrid, Spain), and the resultant peaks were detected by measuring its absorbance at 280 nm analyzed with EMPOWER software (Waters, Santa Clara, CA, USA). We used the Yust method to determine the tryptophan content [17 (link)].