A BRANCHED MONOMETHOXYPOLY(ETHYLENE GLYCOL) FOR PROTEIN MODIFICATION

Procedures are described for linking monomethoxypoly(ethylene glycol) (mPEG) to both epsilon and alpha amino groups of lysine. The lysine ca rboxyl group can then be activated as a succinimidyl ester to obtain a new mPEG derivative (mPEG2-COOSu) with improved properties for biotec hnical applications. This branched reagent showed in some cases a lowe r reactivity toward protein amino groups than the linear mPEG from whi ch it was derived. A comparison of mPEG- and mPEG2-modified enzymes (r ibonuclease, catalase, asparaginase, trypsin) was carried out for acti vity, pH and temperature stability, K-m and K-cat values, and protecti on to proteolytic digestion. Most of the adducts from mPEG and mPEG2 m odification presented similar activity and stability toward temperatur e change and pH change, although in a few cases mPEG2 modification was found to increase temperature stability and to widen the range of pH stability of the adducts. On the other hand, all of the enzymes modifi ed with the branched polymer presented greater stability to proteolyti c digestion relative to those modified with the linear mPEG. A further advantage of this branched mPEG lies in the possibility of a precise evaluation of the number of polymer molecules bound to the proteins; u pon acid hydrolysis, each molecule of mPEG2 releases a molecule of lys ine which can be detected by amino acid analysis. Finally, dimerizatio n of mPEG by coupling to lysine provides a needed route to monofunctio nal PEGs of high molecular weight.