Hydrodynamic behavior and thermal stability of a PEGylated protein: Studies with hen egg lysozyme

<p>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.</p><p>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 (&amp;sim;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 &amp;sim;50 A upon modification with a single 20-kD PEG chain. PEG2-HEL and PEG3-HEL had even larger radii of &amp;sim;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 &amp;sim;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.</p><p>The thermal stability of PEGylated HEL was evaluated by employing the Eyring-Lumry model ( N&amp;harr;TmD &amp;rarr;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&amp;deg;C (DSC) and 4.0&amp;deg;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 increasing degree of PEGylation; only 34% of the HEL monomer remained after incubation at 75&amp;deg;C for 30 minutes, while 68% and 79% of the PEG1-HEL and PEG2-HEL monomers were present. These data suggest that the thermal stability of PEGylated HEL is kinetically controlled. The Tm may not be a true indicator of the stability of a PEG-protein with respect to aggregation.</p><p>1Do---Diffusion coefficient value extrapolated to infinite dilution. 2N---native state, D---denatured state, A---aggregate.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Dissertation

Year

2003

Author dc:creator

• Gokarn, Yatin R

Contributors dc:contributor

• Thomas M Lane

• Biophysics
• General
• Chemistry
• Biochemistry
• Health Sciences
• Pharmacy

Repository record dc:identifier

https://scholars.unh.edu/dissertation/192

OAI identifier oai:identifier

oai:scholars.unh.edu:dissertation-1191