Investigation of changes in recombinant human interleukin-2 structure in the presence of functionalized-glucose additives in solution

Date of Completion

January 2000

Keywords

Health Sciences, Pharmacology|Biophysics, General

Degree

Ph.D.

Abstract

Functionalized-glucose polymers such as sodium carboxymethylcellulose, hyaluronic acid, and chitosan are currently being investigated for use in protein formulations. It is necessary to understand the effect of these polymers on the structure of the protein. Therefore, interactions between functionalized-glucose polymers and recombinant human interleukin-2 (rhIL-2) were investigated. ^ Interactions between functionalized-glucose molecules and rhIL-2 were investigated by measuring the tryptophan fluorescence, aggregation and extrinsic fluorescence studies. Thermal denaturation experiments were done to evaluate the susceptibility of the protein to unfold in the presence of these molecules. The presence of the polymer resulted in unfolding of the protein at low temperatures. To further understand the specific effect of these polymers on the structure of proteins, small molecules glucuronic acid, glucose and glucosamine that resemble the monomer units of these polymers were used. The presence of glucuronic acid caused rhIL-2 aggregation. Addition of glucosamine and glucose resulted in an irreversible change to the protein structure during thermal denaturation. The presence of glucuronic acid resulted in reversible change to the structure. Thermally induced rhIL-2 aggregation was enhanced in rhIL-2-glucosamine and rhIL-2-glucose samples. Presence of glucuronic acid resulted in limiting thermally induced aggregation. The effect of glucuronic acid on rhIL-2 structure was determined to be electrostatic in nature, which can result in limiting the thermal denaturation and aggregation of the protein. ^ The surface hydrophobicity of the protein did not change in the presence of glucosamine and glucose while presence of glucuronic acid (200μM) resulted in changes in surface hydrophobicity due to change in rhIL-2 structure. Glucuronic acid (greater than 30mM) resulted in changes to the far-UV circular dichroism (FUV-CD) signal indicating changes to the protein secondary structure. Near-UV CD studies showed changes to the tertiary structure of the protein when glucuronic acid (120μM) was present which in turn resulted in increased surface hydrophobicity of the protein. Dialysis was performed to study the reversibility of changes to the rhIL-2 structure. Removal of glucuronic acid (60mM) from samples that were heated from 15°C to 75°C and cooled back to 15°C resulted in reversal of these structural changes to the structure seen for native rhIL-2. Thermally induced changes in rhIL-2 were found to be irreversible when glucosamine and glucose were present in solution. The presence of glucuronic acid (60mM) resulted in reversible structural change to the protein structure. ^

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