RP HPLC binding domains of proteins

Procedures have been developed to identify the chromatographic binding doma ins of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) dur ing their interaction with reversed-phase sorbent materials. The procedure involves adsorption of the protein solute to the chromatographic sorbent, f ollowed by proteolytic cleavage. Comparison of the proteolytic map obtained for Cyt c and bGH in free solution with the corresponding map obtained whe n these proteins are adsorbed to the chromatographic sorbent revealed signi ficant differences in the digestion pattern. Following characterization of the peptides generated in both maps, the results indicated that specific re gions on the surface of both Cyt c and bGH are inaccessible to tryptic clea vage when adsorbed to the hydrophobic surface of both a C-4 and a C-18 sorb ent. Based on the assumption that the region of the protein surface that is in contact with the sorbent remains intact and bound to the sorbent during the digestion step, while the protein surface that is exposed to the solve nt is accessible to proteolysis, the regions that were inaccessible to tryp tic digestion were found to correspond to hydrophobic domains on the protei n surface. These results also suggest that the three-dimensional structures of these proteins remain largely intact upon adsorption to the hydrophobic surface.