Prostar 215

SEC experiments were carried out on a Varian ProStar 215 solvent delivery system, coupled to a Varian ProStar 320 UV/VIS detector set at a wavelength of 280 nm. Separation was performed using a BioSep 5-μm SEC-s2000 145 Å, 300 × 4.6-mm LC column (Phenomenex, Torrance, CA). 50 mm sodium phosphate buffer, pH 6.65, containing 150 mm NaCl was used as eluent buffer. Flow rate was set at 0.5 ml/min. Proteins were injected at 0.1 mg/ml concentration in the presence or absence of 1 mg/ml low-molecular-mass heparin (commercial name Fragmin®, Pfizer). Molecular masses of proteins were calculated using the calibration curve provided by the manufacturer.

First, SEC experiments were carried out on a Varian ProStar 215 solvent delivery system coupled to a Varian ProStar 320 UV-visible detector. Separation was performed using BioSep 5 μm SEC-s2000 145 Å, 300 × 4.6 mm LC column (Phenomenex, Torrance, CA, USA). 50 mm sodium phosphate buffer, pH 6.65, containing 150 mm NaCl was used as an eluent at a 0.5 ml/min flow rate. met–Evasin-4 was injected at 0.1 mg/ml concentration and followed by absorbance at λ = 280 nm.
In addition, SEC-MALS was used to determine the oligomeric state of Evasin-4 by injecting 100 μl of protein at 5.3 mg/ml onto a Superdex 75 10/300 gel filtration column (GE Healthcare) in 20 mm Tris, 100 mm NaCl, pH 7.3, at 17 °C. SEC was performed with online static light scattering (miniDAWN TREOS, Wyatt Technology) and differential refractive index determination (Shimadzu RID-10A) on a Shimadzu HPLC system. Protein concentration at elution and massed average molecular mass was determined using standard protocols in the ASTRA6 program (Wyatt). The set-up was calibrated with 100 μl of 5 mg/ml monomeric chicken egg white albumin (Sigma–Aldrich). A refractive index increment (dn/dc) value of 0.185 ml/g was used for Evasin-4.