AbstractProtein conjugation with polyethylene glycol (PEG) is a valuable means for improving stability, solubility and bioavailability of pharmaceutical proteins. Using human galectin-2 (hGal-2) and 5 kDa PEG as a model system we first produced a PEG-hGal-2 conjugate exclusively at the Cys75 residue, resulting in two mono-substituted subunits per hGal-2 homodimer. Small angle x-ray and neutron scattering (SAXS and SANS) were combined to provide complementary structural information about the PEG-hGal-2 conjugate, wherein signal generation in SAXS depends mainly on the protein while SANS data presents signals from both the protein and PEG moieties. SAXS data gave a constant radius of gyration (Rg = 21.5 Å) for the conjugate at different concentrations, and provided no evidence for an alteration of homodimeric structure or hGal-2 ellipsoidal shape upon PEGylation. In contrast, SANS data revealed a concentration dependence of Rg for the conjugate, with the value decreasing from 31.5 Å at 2 mg/mL to 26 Å at 14 mg/mL (based on hGal-2 concentration). Scattering data have been successfully described by model of ellipsoidal homogeneous core (hGal-2) attached with polymer chains (PEG) at surface. Evidently, the PEG conformation of conjugate strongly depends on the conjugate concentration and PEG’s radius of gyration decreases from 24.5 to 15 Å. An excluded volume effect, arising from steric clashes between PEG molecules at high concentration, was quantified by estimating the second virial coefficient, A2, of PEGylated hGal-2 from the SANS data. A positive value of A2 (6.0±0.4 ×10-4 cm3 mol g-2) indicates repulsive interactions between molecules, which are expected to protect protein against aggregation.