PROTEIN-TRANSPORT ACROSS HYDRATED HYALURONIC-ACID ESTER MEMBRANES - EVALUATION OF RIBONUCLEASE-A AS A POTENTIALLY USEFUL MODEL PROTEIN

The study of mechanisms governing release of proteins and peptides fro m polymeric matrices is often complicated by structural instability co mmonly associated with exposure of proteins to conditions used during matrix incorporation and subsequent release studies. The purpose of th e present work was to investigate ribonuclease A (RNase A) as a potent ial model protein for probing mechanisms of protein release from matri ces composed of partially esterified hyaluronic acid. The aqueous stab ility of RNase A and structural recovery following exposure to organic solvent were evaluated using a variety of analytical techniques, and the permeability of intact RNase A through partially esterified hyalur onic acid membranes was determined. RNase A showed excellent aqueous s tability and permeated hyaluronate membranes with no apparent changes in protein size. Also, while CD studies showed significant structural alteration of RNase A dissolved in an organic solvent, this alteration was largely reversible upon evaporation of the solvent and dissolutio n of the protein residue in aqueous buffer. The biological activity of RNase A was maintained following diffusion through the polymer matrix and release from protein-loaded membranes. Permeability of RNase A th rough the partially esterified hyaluronic acid membranes was nonlinear ly dependent on the degree of polymer esterification, and diffusion be havior of the protein in the hydrated polymer membranes was consistent with Yasuda's free volume theory.