RP-HPLC binding domains of proteins.
Procedures have been developed to identify the chromatographic binding domains of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) during their interaction with reversed-phase sorbent materials. The procedure involves adsorption of the protein solute to the chromatographic sorbent, followed by proteolytic cleavage. Comparison of the proteolytic map obtained for Cyt c and bGH in free solution with the corresponding map obtained when these proteins are adsorbed to the chromatographic sorbent revealed significant differences in the digestion pattern. Following characterization of the peptides generated in both maps, the results indicated that specific regions on the surface of both Cyt c and bGH are inaccessible to tryptic cleavage when adsorbed to the hydrophobic surface of both a C-4 and a C-18 sorbent. 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 solvent is accessible to proteolysis, the regions that were inaccessible to tryptic digestion were found to correspond to hydrophobic domains on the protein surface. These results also suggest that the three-dimensional structures of these proteins remain largely intact upon adsorption to the hydrophobic surface.
Aguilar
M I
MI
Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia. mibel.aguilar@med.monash.edu.au
Clayton
D J
DJ
Holt
P
P
Kronina
V
V
Boysen
R I
RI
Purcell
A W
AW
Hearn
M T
MT
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D013006:Growth Hormone / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D003574:Cytochrome c Group; D010446:Peptide Fragments; D013006:Growth Hormone; D014357:Trypsin