Biochemical and biophysical characterization of lysozyme modified by PEGylation

Abstract

PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH(2) modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FTIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 degrees C, and demonstrated resistance to proteolytic degradation.

Evaluation of in vitro cytotoxic activity of mono-PEGylated StAP3 (Solanum tuberosum aspartic protease 3) forms

“…The apparent molecular weight of the different PEGylated species obtained from size exclusion chromatography and gel electrophoresis (SDS-PAGE) is overestimated due to the retarded mobility of PEGylated proteins, which has been previously reported [56], [57]. Moreover, it has also been reported that a 5 kDa-PEG-conjugated protein increases its apparent molecular weight in 15 kDa approximately [58]. This phenomenon has been attributed to the fact that the hydrodynamic volume for a PEG-conjugated protein results higher than the expected for a protein of similar molecular weight, due to the high hydrophilicity of the PEG unit [59], [60].

Fig.

…”

Pentafluorophenyl Ester-Functionalized Phosphorylcholine Polymers: Preparation of Linear, Two-Arm, and Grafted Polymer–Protein Conjugates

“…PEGylated G-CSF (Neulasta), for example, retains 41% of its native enzymatic activity. 11 Prior studies with lysozyme conjugated to a 5 kDa PEG chain showed suppressed enzymatic activity (77%) relative to the native enzyme, 49 whereas a study on polyMPC-BSA and lysozyme conjugates showed 75−90% of native enzymatic activity. 26 These data suggest that polymer conjugation does not markedly reduce enzyme activity but that a modest reduction in activity may result from polymer shielding effects (steric interference).…”

Milling solid proteins to enhance activity after melt-encapsulation

“…This effect can be mitigated via chemical modification of the protein, for example the covalent attachment of a hydrophobic dye or the polymer polyethylene glycol (PEG) (Lee et al, 2015). However, the reaction products can be difficult to separate, require extensive filtration or chromatography to remove unreacted components, and result in substantial purification loss (Freitas and Abrahão-Neto, 2010; Lee et al, 2003). One of the advantages of utilizing HME as an alternative to solvent based encapsulation methods is the increased encapsulation efficiency, scale-up potential, and solventless nature (Maniruzzaman et al, 2012; Repka et al, 2012).…”