Improved activity and stability of lysozyme at the water/CH2Cl2 interface: enzyme unfolding and aggregation and its prevention by polyols.
Protein inactivation and aggregation at the water/CH2Cl2 interface is one of the most detrimental events hindering the encapsulation of structurally unperturbed proteins into poly(lactide-co-glycolide) (PLG) microspheres for their clinical application as sustained release dosage forms. We have investigated the inactivation and aggregation of the model protein hen egg-white lysozyme at this interface and devised methods to prevent both events. When lysozyme was exposed to a large water/CH2Cl2 interface achieved by homogenization, lysozyme aggregation occurred. Fourier-transform infrared (FTIR) spectroscopic data demonstrated that the aggregates formed contained intermolecular beta-sheets. The aggregates were of a noncovalent nature because they slowly dissolved in D2O and the IR spectral bands typical for the intermolecular beta-sheets disappeared at approximately 1617 and 1690 cm(-1). The observed loss in specific enzyme activity of soluble lysozyme was caused by the irreversible formation of an unfolded lysozyme species, which was found to be monomeric, and was able to leave the water/CH2Cl2 interface and accumulate in the aqueous phase. Polyols were, in a concentration dependent fashion, efficient in ameliorating lysozyme unfolding and aggregation. However, prevention of lysozyme aggregation and activity loss in the various samples were unrelated. Thus, polyols must work by more than one mechanism preventing the two events. For the first time, an excipient effect on the conformational stability of lysozyme has been excluded from contributing to the prevention of lysozyme unfolding and aggregation.
Pérez
C
C
Department of Chemistry, University of Puerto Rico, San Juan 00931-3346, USA.
Griebenow
K
K
D003692:Delayed-Action Preparations / Q000737:chemistry*; D008752:Methylene Chloride / Q000737:chemistry; D008863:Microspheres; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D011098:Polyglactin 910 / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry
D003692:Delayed-Action Preparations; D011108:Polymers; D011506:Proteins; D012997:Solvents; C024617:polyol; D014867:Water; D011098:Polyglactin 910; D008752:Methylene Chloride; C421864:hen egg lysozyme; D009113:Muramidase