Protein and polypeptide orientation on charged surfaces
Abstract
The mechanism by which polypeptides and proteins interact at a liquid/solid interface was investigated utilizing N-hydroxysuccinimide activated succinic acid (SA) as a coupling agent. In this work, adsorption of the small peptide GHRP, ACTH 11-24, cytochrome c and chicken egg white Lysozyme (CEWL) have been examined. A system was designed which allowed multiple binding sites and exact contact regions to be observed through ester and amide bond formation between amine residues on polypeptides and charged, activated surfaces. Interacting bound sites on a polypeptide, locations of the interacting sites in the 3-D structure of the peptide, specific nucleophilicity of the lysyl amino acid side chains on these polypeptides, and orientation of proteins on anionic surfaces was examined as a function of pH. CEWL has 6 lysyl chains on the surface (1-2) of which 2 were specifically implicated in binding to the anionic test surface. It was discovered that with cytochrome c that a specific lysine cluster was the site responsible for electrostatic adsorption and binding. Peptides sequenced at these sites were identified by MALDI/TOF/MS, tandem MS, and Edman sequencing. The results of these experiments have given new understanding to the chromatographic behavior of proteins relative to the number of electrostatic interactions between a protein and ionic stationary phases. This study provides the first chemical evidence that regio-specific orientation of proteins occurs during adsorption on charged stationary phases.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Organic chemistry|Biochemistry|Chemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.