Hydrodynamic behavior and thermal stability of a PEGylated protein: Studies with hen egg lysozyme
type: CreativeWork
description: We studied the effect of covalent attachment of polyethylene glycol (PEGylation) on the hydrodynamic behavior and thermal stability of a model protein---Hen Egg Lysozyme (HEL). HEL was modified with a linear, 20-kD, PEG to produce mono (PEG1-HEL), di (PEG2-HEL), and triPEGylated (PEG3-HEL) species. The hydrodynamic properties of HEL were altered upon PEGylation. A decrease in sedimentation (s) and diffusion (D) coefficients was observed for all three PEG-HEL molecules in comparison to HEL (1.81 s). Despite differences in molecular weights of the PEG-HEL molecules (∼34, 55 and 80 kD), their s values were very close (1.0--1.1 s). Significant hydrodynamic non-ideality was observed for the PEG-HEL molecules, however, their Stokes radii (Rh) calculated from Do1 values were in agreement with dynamic light scattering (DLS) measurements. The Rh of HEL increased dramatically from 20 A to ∼50 A upon modification with a single 20-kD PEG chain. PEG2-HEL and PEG3-HEL had even larger radii of ∼68 A and 74 A. DLS studies with various PEGS (MW 5000--40,000) indicated that PEG is a random coil in solution. The Rh of PEG1-HEL and PEG2-HEL were measured to be only ∼10% larger than the 20-kD (43 A) and 40-kD (60 A) PEG chains. These data suggest that the covalently tethered PEG(s) predominantly govern the solution conformation of the PEG-HEL molecules. The thermal stability of PEGylated HEL was evaluated by employing the Eyring-Lumry model ( N↔TmD →kaA )2 for protein aggregation. A decrease in the melting temperature (Tm) of HEL unfolding was observed with increasing degree of PEGylation, which is indicative of thermodynamic instability. A Tm drop of up to 2.5°C (DSC) and 4.0°C (Difference Spectrum method) was observed for the PEG-HEL molecules. In contrast, turbidimetric studies showed that the kinetic aggregation rate (ka) of the PEG-HEL molecules was dramatically lower in comparison to the native HEL. Size exclusion HPLC indicated that the extent of aggregation decreased with increa…
abstract: We studied the effect of covalent attachment of polyethylene glycol (PEGylation) on the hydrodynamic behavior and thermal stability of a model protein---Hen Egg Lysozyme (HEL). HEL was modified with a linear, 20-kD, PEG to produce mono (PEG1-HEL), di (PEG2-HEL), and triPEGylated (PEG3-HEL) species. The hydrodynamic properties of HEL were altered upon PEGylation. A decrease in sedimentation (s) and diffusion (D) coefficients was observed for all three PEG-HEL molecules in comparison to HEL (1.81 s). Despite differences in molecular weights of the PEG-HEL molecules (∼34, 55 and 80 kD), their s values were very close (1.0--1.1 s). Significant hydrodynamic non-ideality was observed for the PEG-HEL molecules, however, their Stokes radii (Rh) calculated from Do1 values were in agreement with dynamic light scattering (DLS) measurements. The Rh of HEL increased dramatically from 20 A to ∼50 A upon modification with a single 20-kD PEG chain. PEG2-HEL and PEG3-HEL had even larger radii of ∼68 A and 74 A. DLS studies with various PEGS (MW 5000--40,000) indicated that PEG is a random coil in solution. The Rh of PEG1-HEL and PEG2-HEL were measured to be only ∼10% larger than the 20-kD (43 A) and 40-kD (60 A) PEG chains. These data suggest that the covalently tethered PEG(s) predominantly govern the solution conformation of the PEG-HEL molecules. The thermal stability of PEGylated HEL was evaluated by employing the Eyring-Lumry model ( N↔TmD →kaA )2 for protein aggregation. A decrease in the melting temperature (Tm) of HEL unfolding was observed with increasing degree of PEGylation, which is indicative of thermodynamic instability. A Tm drop of up to 2.5°C (DSC) and 4.0°C (Difference Spectrum method) was observed for the PEG-HEL molecules. In contrast, turbidimetric studies showed that the kinetic aggregation rate (ka) of the PEG-HEL molecules was dramatically lower in comparison to the native HEL. Size exclusion HPLC indicated that the extent of aggregation decreased with increa…
author: Gokarn, Yatin R
keywords: Biophysics, General, Chemistry, Biochemistry, Health Sciences, Pharmacy
date published: 2003-01-01T08:00:00Z
date modified: 2026-07-24T05:22:01Z
provider: University of New Hampshire
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of New Hampshire