1998 Volume 13 Issue 3 Pages 173-178
Brain-derived neurotrophic factor (BDNF) was modified by carboxyl-directed protein pegylation in order to retain biologic activity and reduce the systemic clearance of this cationic protein in vivo. Since the modification of the surface lysine residue of BDNF resulted in loss of biologic activity, the present study examine the feasibility of placing polyethylene glycol (PEG) polymer on the carboxyl residue of BDNF, PEG molecules with terminal hydrazide moiety of molecular weight 2 kDa (PEG2000-Hz) and 5 kDa (PEG5000-Hz) were coupled to BDNF caboxyls using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide. Polyacrylamide gel electrophoresis revealed that the molecular weight distributions of BDNF-PEG2000 and BDNF-PEG5000 were 20 k∼30 kDa and 60 k∼80 kDa, respectively. Cell survival studies using 3T3 cells transfected with the BDNF receptor gene showed that the biologic activity of BDNF was not changed following pegylation with PEG2000, and was minimally impaired following pegylation with PEG5000. The systemic clearances of the 125I-BDNF-PEG2000 and 125I-BDNF-PEG5000 following intravenous administration to rats were reduced 67% and 91%, respectively, compared to unconjugated BDNF. TCA soluble fraction of the radioactivity in the plasma 60 min following administration were reduced 72% and 94%, respectively, showing the significant reduction of the BDNF metabolism by pegylation. These experiments describe the first successful carboxyl-directed pegylation of a neuropeptide, and show that this formulation substantially reduces the systemic clerance and metabolism of this neurotrophic factor. In conclusion, the carboxyl-directed pegylation is beneficial to the modification of the protein whose surface lysine or arginine residue is essential to the biologic activity.
