Effect of chemical modification of recombinant human acetylcholinesterase by polyethylene glycol on its circulatory longevity

Post-translational modifications were recently shown to be responsible for the short circulatory mean residence time (MRT) of recombinant human acetyl eholinesterase (rHuAChE) [Kronman, Velan, Marcus, Ordentlich, Reuveny and S hafferman (1995) Biochem. J. 311, 959-967; Chitlaru, Kronman, Zeevi, Kam, H arel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647-658; Chi tlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613-625], whic h is one of the major obstacles to the fulfilment of its therapeutic potent ial as a bioscavenger. In the present study we demonstrate that the MRT of rHuAChE can be significantly increased by the controlled attachment of poly ethylene glycol (PEG) side chains to lysine residues. Attachment of as many as four PEG molecules to monomeric rHuAChE had minimal effects, if any, on either the catalytic activity (K-m = 0.09 mM and k(cat) = 3.9 x 10(5) min( -1)) or the reactivity of the modified enzyme towards active-centre inhibit ors, such as edrophonium and di-isopropyl fluorophosphate, or to peripheral -site ligands, such as propidium, BW284C51 and even the bulky snake-venom t oxin fasciculin-II. The increase in MRT of the PEG-modified monomeric enzym e is linearly dependent, in the tested range, on the number of attached PEG molecules, as well as on their size. It appears that even low level PEG-co njugation can overcome the deleterious effect of under-sialylation on the p harmacokinetic performance of rHuAChE. At the highest tested ratio of attac hed PEG-20000/rHuAChE (4:1), an MRT of over 2100 min was attained, a value unmatched by any other known form of recombinant or native serum-derived AC hE reported to date.