Influence of lysozyme complexation with purified Aldrich humic acid on lysozyme activity

Abstract

Humic acid is an important component of dissolved organic matter and in two previous papers it has been shown that purified Aldrich humic acid (PAHA) forms strong complexes with the oppositely charged protein lysozyme (LSZ). The complexation and aggregation of enzymes with humic acids may lead to changes in their activity. Therefore, the enzyme activity of LSZ in the PAHA – LSZ complex as a function of mass ratio PAHA:LSZ was investigated at two pH values (pH 5 and pH 8 where PAHA and LSZ are oppositely charged) and two ionic strengths (5 mmol l−1; 50 mmol l−1). The newly prepared complex was characterized and the mass ratios PAHA:LSZ at the isoelectric point (IEP) of the complex at the assayed conditions were obtained with the Mütek particle charge detector. The LSZ activity was measured with an assay specific for LSZ. The activity of LSZ decreased upon complex formation with PAHA and the PAHA effect on the enzyme activity was increased by the subsequent aggregation. A critical PAHA:LSZ mass ratio for the behaviour of the enzyme activity was that at the IEP of the complex. Before this mass ratio was reached the LSZ activity strongly decreased under all conditions. Beyond this mass ratio both pH and ionic strength affected the activity of LSZ in the complex. The results can be explained by the effects that pH and ionic strength have on (i) the electrostatic attraction between LSZ and PAHA in the complex and (ii) the electrostatic repulsion between the complexes. These two factors plus the hydrophobic attraction determine the structure of the complexes and the size of the aggregates and hence the screening of the active site of the protein.