Complex coacervation of lysozyme and heparin: complex characterization and protein stability

• Published in: Pharmaceutical research 2004-12, Vol.21 (12), p.2354-2359
• Main Authors: van de Weert, Marco, Andersen, Mia Bendix, Frokjaer, Sven
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites - physiology; Chickens; Heparin - chemistry; Heparin - metabolism; Muramidase - chemistry; Muramidase - metabolism; Protein Binding - physiology; Swine
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-004-7689-z

To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability. Insoluble lysozyme-heparin complexes were formed at pH 7.2 and the binding stoichiometry determined using a solution depletion method. Protein structure was determined by infrared spectroscopy and intrinsic fluorescence. Protein stability was evaluated using differential scanning calorimetry and followed in a 12-weeks storage stability study at 37 degrees C. Binding stoichiometry between heparin and lysozyme was found to be dependent on ionic strength of the solution. At low ionic strength (I approximately 0.01) about 11 lysozyme molecules could bind to a 17 kDa heparin chain, 3 to a 6 kDa chain, and less than 2 to a 3 kDa chain. At higher ionic strength (I approximately 0.1), only 7 lysozyme molecules could bind to a 18 kDa heparin chain.. Above ionic strengths of approximately 0.32 M, no insoluble complexes were observed. Infrared spectroscopy and intrinsic fluorescence did not show any major changes in protein structure upon complexation to heparin. In contrast, differential scanning calorimetry showed a large decrease in the melting temperature of the protein, from 77 degrees C to 61 degrees C. Moreover, after 12-weeks storage at 37 degrees C, only 60% protein recovery was observed for the complexes, with no loss of protein for the uncomplexed protein. Heparin has multiple binding sites for lysozyme, amounting to at most one lysozyme molecule per 3 disaccharide units of heparin. Complexation decreased lysozyme stability, suggesting that heparin has a higher affinity for the unfolded state than the native state. Similar destabilization may occur for other proteins upon interaction with highly charged polymeric compounds or surfaces.