Interplay of Secondary Structure and Charge on the Diffusion of a Polypeptide through Negatively Charged Aqueous Pores

Abstract

This study was conducted to investigate the interplay of secondary structure and charge of a polypeptide on its permeability through negatively charged pores of synthetic porous membranes and Caco-2 cell monolayers. Poly(D-glutamic acid) [Poly(D-Glu)] was used as a model polypeptide. Transport studies were conducted at 37 degrees C through both track-etched polycarbonate membranes (using side-by-side diffusion cells) and Caco-2 cell monolayers. Apparent permeability coefficients and diffusion coefficients were calculated. When diffusion was unhindered, poly(D-Glu) appeared to be transported at the same rate regardless of whether it existed in the random coil or the alpha-helix secondary structure. When moderately hindered diffusion was evaluated, poly(D-Glu) with partial alpha-helix secondary structure, exhibited significantly greater transport than when the polypeptide predominantly existed as the highly negatively charged random coil. This trend was reversed when the diffusion was severely hindered by the tight junctions of the Caco-2 cell monolayers. Neither charge, nor secondary structure, played a significant role in the unhindered diffusion of poly(D-Glu). When the molecules were moderately hindered, polypeptide/membrane charge interactions significantly influenced the rate of aqueous diffusion. As the overall molecular dimensions of the polypeptide approached the pore size, the inherent molecular flexibility of the random coil secondary structure overcame the effect of charge repulsion.