Abstract
Purpose. To isolate mono-PEGylated epidermal growth factor (EGF) isoforms, identify the site of PEGylation, and evaluate the biologic activity of each isoform. Methods. EGF was PEGylated with an NHS-PEG derivative (Mw 3,400). Mono-PEGylated EGF fraction was separated by gel-filtration HPLC and three mono-PEGylated EGF isoforms were purified by RP-HPLC. Tryptic digestion mapping of both EGF and mono-PEGylated EGF isoforms was performed to identify the PEGylation sites using RP-HPLC. The digested fragments were also analyzed by matrix-assisted laser desorption and ionization time of flight (MALDI-TOF) mass spectroscopy for further verification of the three PEG conjugation sites. The biologic activity of positional isoforms was evaluated by a cell proliferation assay and a receptor tyrosine kinase activity assay to determine the effect of PEGylation site on its activity. Results. Mono-PEGylated EGF was composed of three positional isomers. Tryptic digestion mapping and MALDI-TOF analysis permitted the identification of the PEGylated site of the three isoforms at N-terminus, Lysine 28, and Lysine 48. PEG-N-terminus EGF, among the three positional isomers, showed the highest activity in a cell proliferation assay and in a receptor-binding assay. Conclusion. This study demonstrates that biologic activities of mono-PEGylated EGF isomers are highly dependent upon the site of PEGylation in EGF.
| Original language | English |
|---|---|
| Pages (from-to) | 845-851 |
| Number of pages | 7 |
| Journal | Pharmaceutical Research: An Official Journal of the American Association of Pharmaceutical Scientists |
| Volume | 19 |
| Issue number | 6 |
| DOIs | |
| State | Published - 2002 |
| Externally published | Yes |
Funding
This work was supported by the Center for Advanced Functional Polymers, KAIST, Korea.
| Funders | Funder number |
|---|---|
| Korea Advanced Institute of Science and Technology |
Keywords
- Biologic activity
- Epidermal growth factor (EGF)
- Peptide mapping
- Polyethylene glycol (PEG)