Noncovalent Complexes between Poly(ethylene glycol) and Proteins

I. N. Topchieva^1*, E. M. Sorokina², N. V. Efremova², and A. L. Ksenofontov³

¹School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-0174

²Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia; fax: (095) 954-2732

³Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181

^* To whom correspondence should be addressed.

Received February 11, 1998; Revision received June 5, 1998

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A new method of formation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumin) under high pressure has been developed. The existence of polymer in complexes was proved using ³H-labeled PEG. Complexes between PEG and ChT were studied in detail. It was shown that the composition of complexes (the number of polymer chains per ChT molecule) depends on the molecular mass of PEG and decreases with the increase of molecular mass from 300 to 4000. At the same time, the portion of the protein (wt. %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to ~70 wt. %. It was shown that kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG--ChT complexes are identical to the corresponding values for the native ChT. The conformational properties of ChT in complexes were studied by circular dichroism. It was shown that the enzyme in complexes fully retains its secondary structure. The estimation of steric availability of PEG polymer chains in complexes was evaluated by the complexation with alpha-cyclodextrin (CyD). It was shown that in contrast to free PEG, only part (~10%) of PEG polymer chains in PEG--ChT complexes participate in the complexation with CyD. Hence, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule in a region far from the active site of the enzyme and results in the significant decrease in the mobility of polymer chains.

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KEY WORDS: alpha-chymotrypsin, poly(ethylene glycol), polymer--protein complexes, high pressure, enzymatic properties, circular dichroism, sedimentation

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