Fig. 2.

TGF-β₂ is bound to chondroitin sulfate proteoglycans in milk. A: we immunoprecipitated TGF-β₂ and resolved the immune complexes in nondenaturing gels. Staining with coomassie blue (top) or alcian blue (middle) dyes showed a prominent 110-kDa band. The same band also showed immunoreactivity for chondroitin sulfate. Data represent preterm milk samples from four mothers, tested in at least three separate runs. B: computational model predicts the binding of a chondroitin sulfate hexasaccharide sequence to TGF-β₂ at two potential binding sites (Site 1, Site 2). Chondroitin sulfate sequence is depicted in a sticks form, whereas TGF-β₂ is depicted in grey ribbons with individual residues predicted to be involved in some interaction at an atomic level in ball and stick form. Helices are denoted as hH₁, hH₂, and hH₃. Site 1 residues include Lys25, Lys37, Glu35, and Tyr91 and Site 2 residues include His58, Ser80, Asn103, Lys110, and Ser112. Dotted black box depicts the possible orientation of intervening chondroitin sulfate residues connecting the reducing end (RE) of Site 2-bound chain to the nonreducing end (NRE) of Site 1-bound chondroitin sulfate chain. C: affinity capillary electrophoresis demonstrates biophysical interaction between chondroitin sulfate and TGF-β₂. Panel shows electropherogram of 0.5 μM TGF-β₂ in sodium phosphate buffer containing different concentrations of chondroitin sulfate. Dimethyl sulfoxide is the marker. Data represent findings from three separate experiments. D: binding curve of chondroitin sulfate-TGF-β₂ system, fitted for a one-site total binding equation.