Torchilin V P, Maksimenko A V, Smirnov V N, Berezin I V, Klibanov A M, Martinek K
Biochim Biophys Acta. 1979 Mar 16;567(1):1-11. doi: 10.1016/0005-2744(79)90165-7.
The dependence of alpha-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermoinactivation (50 degrees C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4, NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.
研究了α-胰凝乳蛋白酶的热稳定性和催化活性对其氨基修饰程度的依赖性。通过烷基化（使用丙烯醛，随后用硼氢化钠还原席夫碱）和酰化（使用琥珀酸酐或乙酸酐）进行修饰。已确定，大多数被滴定氨基（约80%）的修饰对表征酶热失活单分子过程（50℃，pH 8）的一级速率常数只有轻微影响。仅当修饰程度高于80%时，热稳定性才会急剧增加（增加120倍），然而，所有被滴定氨基的完全取代再次导致酶不稳定。得出的结论是，在大约十五个被滴定的氨基中，只有一两个氨基的修饰在酶热稳定性的改变中起关键作用。研究了相对于添加盐（Na2SO4、NaCl、KCl、CCl3COOK、(CH3)4NBr）的性质和浓度的稳定化效果程度。还就其NH2-基团的烷基化程度阐明了胰凝乳蛋白酶的紫外吸收（280nm）。所获得的数据使得可以得出结论，酶修饰导致球蛋白表面层非静电（疏水）相互作用的减少。结果，形成了对变性（展开）更稳定的蛋白质构象。
