Protein Encapsulation by Humic Substances

Protein encapsulation by natural organic matter is hypothesized to preserve the activity of proteins in terrestrial and aquatic environments. Direct molecular-level evidence for encapsulation of net positively charged proteins lysozyme, trypsin, and ribonuclease A by a diverse set of humic substances (HS) in nanostructured films was collected using a combination of optical waveguide lightmode spectroscopy and quartz crystal microbalance measurements. The results suggest that protein–HS electrostatic attraction drives encapsulation of positively charged lysozyme by a soil humic acid at pH 5 to 8 and by six additional humic and fulvic acids from terrestrial and mixed terrestrial aquatic sources at pH 5 and 6. Encapsulation of trypsin and ribonuclease A, which had negatively charged surface patches under the studied conditions, suggested that localized protein–HS electrostatic repulsion is overcompensated by attractive forces, likely including contributions from the hydrophobic effect. Evidence is provided showing that encapsulation of lysozyme at pH 8 and of ribonuclease A at pH 5 and 6 involved partial disassembly of HA supramolecular associations. This work advances a molecular-level picture of protein encapsulation by HS and presents a novel approach to study the effects of encapsulation on protein enzymatic activity and susceptibility to abiotic and biotic transformations.