Institute of Environment Resource and Soil Fertilizer, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; National Agricultural Experimental Station for Soil Quality, Jiaxing 314000, China.
Physical Chemistry and Soft Matter, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands.
Sci Total Environ. 2021 May 10;768:144858. doi: 10.1016/j.scitotenv.2020.144858. Epub 2021 Jan 5.
Modification of enzyme/protein conformation will affect the activities and functionality of enzymes. Previous studies have shown that the activity of lysozyme (LSZ) in the presence of humic acid (HA) is largely determined by the mass ratio of HA/LSZ (f = mHA/mLSZ), pH and ionic strength. Here the interaction and conformation of LSZ in HA/LSZ-complex/aggregate (HA/LSZ-c/a) were investigated by spectroscopic techniques at (initial) pH 5 and 8 and ionic strength 5 mmol/L. The results indicated a strong interaction between HA and LSZ. Circular dichroism (CD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the helix content reached a minimum at the mass ratio of its iso electric point (IEP) at given initial pH, f. The changes in β-sheet and random coil of HA/LSZ-c/a were opposite with increasing f. The minimum of helix content at f corresponded with the minimum LSZ activity and maximum aggregate size of HA/LSZ-c/a. UV-vis spectra and fluorescence measurements indicated that the amino acid residues (especially for tyrosine) in LSZ were in a more hydrophobic microenvironment before f due to the formation of HA/LSZ-c/a, while were gradually exposed to a more polar microenvironment beyond f with the disaggregation of HA/LSZ-c/a. HA and LSZ interaction caused a more hydrophobic microenvironment for the amino acid residues at initial pH 8. This study improves our understanding of enzyme/protein behavior in the natural environment.
酶/蛋白质构象的改变会影响酶的活性和功能。先前的研究表明，在腐殖酸（HA）存在的情况下，溶菌酶（LSZ）的活性在很大程度上取决于 HA/LSZ 的质量比（f = mHA/mLSZ）、pH 值和离子强度。本研究采用光谱技术，在初始 pH 值为 5 和 8 及离子强度为 5 mmol/L 的条件下，研究了 LSZ 在 HA/LSZ-复合物/聚集体（HA/LSZ-c/a）中的相互作用和构象。结果表明，HA 与 LSZ 之间存在强烈的相互作用。圆二色性（CD）和衰减全反射傅里叶变换红外（ATR-FTIR）光谱表明，在给定初始 pH 值下，当 f 等于其等电点（IEP）时，螺旋含量达到最小值。HA/LSZ-c/a 的β-折叠和无规卷曲的变化随 f 的增加而相反。在 f 处，螺旋含量的最小值对应于 LSZ 活性的最小值和 HA/LSZ-c/a 的聚集体尺寸的最大值。紫外-可见光谱和荧光测量表明，由于 HA/LSZ-c/a 的形成，在 f 之前，LSZ 中的氨基酸残基（特别是酪氨酸）处于更疏水的微环境中，而在 f 之后，随着 HA/LSZ-c/a 的解聚，它们逐渐暴露于更亲水的微环境中。HA 和 LSZ 之间的相互作用导致在初始 pH 值为 8 的情况下，氨基酸残基所处的微环境更加疏水。本研究提高了我们对酶/蛋白质在自然环境中行为的理解。
