Kristiansen A, Vårum K M, Grasdalen H
Department of Biotechnology, The Norwegian University of Science and Technology, Trondheim.
Eur J Biochem. 1998 Jan 15;251(1-2):335-42. doi: 10.1046/j.1432-1327.1998.2510335.x.
We have investigated the binding interactions between highly de-N-acetylated chitosans and lysozyme from chicken egg white by one-dimensional and two-dimensional 1H-NMR spectroscopy. A fully de-N-acetylated chitosan (fraction of N-acetylated units, F < 0.001) induced no observable changes in the 1H chemical shifts of lysozyme. However, a chitosan with F(A) = 0.04, where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated sequence), induced significant shifts of several lysozyme resonances, demonstrating a specific interaction between lysozyme and de-N-acetylated units in the chitosan. The interaction between the two positively charged molecules increased with increasing ionic strength, as expected. The dissociation constant (Kd) between lysozyme and the monoacetylated sequence was strongly dependent on pH* (pH measured in D2O), with Kd = 0.02+/-0.01 mM at pH* 6.0, Kd = 0.11+/-0.02 mM at pH* 4.5, and Kd approximately 2 mM at pH* 3, suggesting that electrostatic forces contribute to the observed binding. The complex was strikingly stable, with bound lifetimes in the range of 10-25 ms at pH* 4.5 and 328-300 K. Most lysozyme resonances that were affected by the binding were assigned, and we suggest that the monoacetylated chitosan sequence binds to the active site cleft of lysozyme with the N-acetylated unit in subsite C. Assuming this binding mode, we have discussed the contributions in energetic terms from individual subsites of lysozyme towards binding of N-acetylated and de-N-acetylated units.
我们通过一维和二维¹H-NMR光谱研究了高度脱N-乙酰化壳聚糖与鸡蛋白溶菌酶之间的结合相互作用。完全脱N-乙酰化的壳聚糖（N-乙酰化单元的比例，F < 0.001）未引起溶菌酶¹H化学位移的可观察到的变化。然而，一种F(A)=0.04的壳聚糖，其中N-乙酰化单元主要被脱N-乙酰化单元包围（单乙酰化序列），引起了几个溶菌酶共振峰的显著位移，表明溶菌酶与壳聚糖中的脱N-乙酰化单元之间存在特异性相互作用。正如预期的那样，两个带正电荷的分子之间的相互作用随着离子强度的增加而增强。溶菌酶与单乙酰化序列之间的解离常数（Kd）强烈依赖于pH*（在D₂O中测量的pH），在pH* 6.0时Kd = 0.02±0.01 mM，在pH* 4.5时Kd = 0.11±0.02 mM，在pH* 3时Kd约为2 mM，这表明静电力有助于观察到的结合。该复合物非常稳定，在pH* 4.5和328 - 300 K时结合寿命在10 - 25 ms范围内。大多数受结合影响的溶菌酶共振峰被归属，并且我们认为单乙酰化壳聚糖序列以亚位点C中的N-乙酰化单元与溶菌酶的活性位点裂隙结合。假设这种结合模式，我们从能量角度讨论了溶菌酶各个亚位点对N-乙酰化和脱N-乙酰化单元结合的贡献。
