Protein Structure Change on Adherence to Ultrafiltration Membranes: An Examination by Electron Paramagnetic Resonance Spectroscopy

Abstract

Electron paramagnetic resonance spectroscopy was used to examine changes in the distance between two spin-labels on hen egg lysozyme (HEL, 45 Å × 30 Å × 30 Å, 14,600 Da) when interacting with membranes during ultrafiltration (UF). Membranes with nominal molecular weight cutoff (MWCO) values ranging from 10,000 to 300,000 Da were used. Both cellulosic (hydrophilic) and polysulfone (hydrophobic) membranes were studied. The technique used is based on spin–spin interaction of protein-bound spin-labels that cause dipolar broadening of the EPR spectra. This line broadening is related to spin-label distance and was subsequently used to infer protein conformational changes. The results demonstrate that the distance between the two labels on HEL, while in aqueous solution, was approximately 14.4 Å. Using the known crystal structure of hen egg lysozyme, molecular computer modeling of the doubly labeled protein suggests that the most probable spin–spin distance in solution is 13.9 Å. This distance is in excellent agreement with our experimentally observed result. Protein–membrane interaction reduced this distance approximately by 5 Å, irrespective of the membrane morphology. It is speculated that the lack of variation seen in all the membranes, under our test conditions, may be due to limitations associated with spin-label locations.