Topchieva I N, Sorokina E M, Efremova N V, Ksenofontov A L
School of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia.
Biochemistry (Mosc). 1998 Nov;63(11):1312-8.
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 approximately 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 (approximately 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.
开发了一种在高压下聚乙二醇（PEG）与蛋白质（α-胰凝乳蛋白酶（ChT）、溶菌酶、牛血清白蛋白）形成非共价复合物的新方法。使用3H标记的PEG证明了复合物中聚合物的存在。对PEG与ChT之间的复合物进行了详细研究。结果表明，复合物的组成（每个ChT分子的聚合物链数）取决于PEG的分子量，并且随着分子量从300增加到4000而减少。同时，复合物中蛋白质的比例（重量%）不取决于掺入的PEG的分子量，约为70重量%。结果表明，PEG-ChT复合物催化N-苯甲酰-L-酪氨酸乙酯和偶氮酪蛋白的酶促水解动力学常数与天然ChT的相应值相同。通过圆二色性研究了复合物中ChT的构象性质。结果表明，复合物中的酶完全保留了其二级结构。通过与α-环糊精（CyD）络合评估了复合物中PEG聚合物链的空间可用性。结果表明，与游离PEG相比，PEG-ChT复合物中只有部分（约10%）的PEG聚合物链参与与CyD的络合。因此，PEG与ChT的络合是通过与远离酶活性位点区域的蛋白质球表面基团的多点相互作用进行的，导致聚合物链的流动性显著降低。
