Conformational modifications of lysozyme caused by interaction with humic acid studied with spectroscopy.

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.