Effects of Coupling Chemistry on the Activity of Poly(ethylene glycol)-Modified Alkaline Phosphatase

Abstract

Proteins modified by covalent coupling to poly(ethylene glycol) are of interest for several biotechnical applications. In the present work we compare the effects of four commonly used coupling methods on alkaline phosphatase activi ty ; the four methods use the PEG tresylate, succinimidyl succinate, cyanuric chloride derivative, or carbonyl diimidazole derivative. All routes give active enzyme, with only the cyanuric chloride route giving significant deactivation; none the less the cyanuric chloride derivative is useful at lower degrees of modification. Examination of the Michaelis-Menten parameters for the cy anuric chloride coupling suggests that the loss of activity from this route results from intramolecular crosslinking of the protein, which in turn leads to loss of protein conformational flexibility.