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 increasing degree of PEGylation; only 34% of the HEL monomer remained after incubation at 75°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.
1Do---Diffusion coefficient value extrapolated to infinite dilution. 2N---native state, D---denatured state, A---aggregate.
","abstract_html":"<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 (&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 &sim;50 A upon modification with a single 20-kD PEG chain. PEG2-HEL and PEG3-HEL had even larger radii of &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 &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&harr;TmD &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&deg;C (DSC) and 4.0&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&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>","abstract_has_math":false,"creators":["Gokarn, Yatin R"],"institution":null,"degree_name":"Doctor of Philosophy","degree_level":"Dissertation","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Thomas M Lane"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2003,"date_issued":"2003-01-01T08:00:00Z","date_published":"2003-01-01T08:00:00Z","updated_at":"2026-07-24T05:22:01Z","subjects":["Biophysics","General","Chemistry","Biochemistry","Health Sciences","Pharmacy"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://scholars.unh.edu/dissertation/192","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Thomas M Lane"]},{"key":"dc:creator","label":"Author","values":["Gokarn, Yatin R"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biophysics","General","Chemistry","Biochemistry","Health Sciences","Pharmacy"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholars.unh.edu/dissertation/192"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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 increasing degree of PEGylation; only 34% of the HEL monomer remained after incubation at 75°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.
1Do---Diffusion coefficient value extrapolated to infinite dilution. 2N---native state, D---denatured state, A---aggregate.
"]},{"key":"dc:title","label":"Title","values":["Hydrodynamic behavior and thermal stability of a PEGylated protein: Studies with hen egg lysozyme"]}]}],"canonical_facts":{"dc:contributor":["Thomas M Lane"],"dc:creator":["Gokarn, Yatin R"],"dc:description.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 increasing degree of PEGylation; only 34% of the HEL monomer remained after incubation at 75°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.
1Do---Diffusion coefficient value extrapolated to infinite dilution. 2N---native state, D---denatured state, A---aggregate.
"],"dc:identifier":["https://scholars.unh.edu/dissertation/192"],"dc:subject":["Biophysics","General","Chemistry","Biochemistry","Health Sciences","Pharmacy"],"dc:title":["Hydrodynamic behavior and thermal stability of a PEGylated protein: Studies with hen egg lysozyme"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy"]},"updated_at":"2026-07-24T05:22:01Z"}