RP-HPLC Binding Domains of Proteins

Abstract

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.

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

“…Adsorption by pipette tips, reaction vials, test and storage tubes, charging devices used in instrumental analysis, tubing and flexible tube connections, extrusion and injection syringes or by chromatographic columns reduces the analyte concentration. It has occasionally been shown to be advantageous to add proteins [10,68,69,70,71], detergents [32,67], or structurally related analogs [72] to the sample solutions to avoid or minimize loss of analyte. In our laboratory we designed a simple quantitative experiment consisting of seven consecutive steps for transfer of a peptide solution from one polypropylene reaction vial (0.5 mL) to another followed by LC-MS quantification.…”

Size Distribution and Amino Acid Chemistry of Base-extractable Proteins from Washington Coast Sediments

“…Although lysine is considered to be relatively hydrophobic and surface active as a free amino acid, lysine typically situates itself on the outside of large globular proteins (Janin 1979;Rose et al 1985). Being on the outside of a globular protein would increase the chance for the residue to be cleaved from parent proteins as an individual amino acid or within a small peptide (Aguilar et al 1998;Nunn et al 2003). Proteases, such as Trypsin, cleave either before or after lysine, thereby increasing the probability of lysine-enriched peptides to occur in the smaller size fractions (see Nunn et al 2003).…”

High‐Performance Liquid Chromatography of Biological Macromolecules

“…The retention process can also result in substantial loss of proteins that become tightly trapped on the separation material, apparently via hydrophobic domains existing in continuous regions on the surfaces of the protein. 233 The recovery of proteins depends largely on chain length of the hydrophobic ligand, with shorter ligands usually affording better yields and conformational integrity. Butyl and cyanopropyl silica are therefore preferred over longer alkyl chains and phenyl groups.…”

“…233 For a sizable molecule such as a protein, the contact functionalities are thus usually those exposed at the protein surface, whereas most of the amino acid residues can take part in the retentive process that involves small peptides. Therefore retention of peptides that have not assumed a secondary structure can be predicted with reasonable accuracy.…”