The Mechanisms Of Soil Humic Acids Interactions With Enzymes

Protein as a component of organic matter, can enter into the soil and water system through the secretion of animals, plants roots and microorganisms, the release from the residue and decomposition of these living things, and human’s activities. Enzymes, e.g. phosphatase and urease, play important roles in the environment, especially for the geochemical cycle of elements such as phosphorus (P), nitrogen (N) etc. Another type of protein like prion causing transmissible spongiform encephalopathies, bacillus thuringiensis (Bt) protein, avian influenza virus, are potentially harmful factors in environment for human and animals. When these proteins are released into environment, they will combine with organic matter, minerals and microorganisms. Humic Substances (HS) are important components of organic matter and are widely distributed in the soil and water. Driven by the electrostatic interaction, hydrophobic force and chemical binding, proteins can be adsorbed onto or entrapped into HS, or can co-polymerize with HS.In this paper, we firstly investigated the colloidal properties of HS and lysozyme (LSZ), especially the surface charge of them. Then we studied the effect of HS on the activity, stability and structure of LSZ. Factors like pH, ionic strength, mass ratio HS/LSZ and types of humic acids were considered. Main results are below:1. We elucidated the method of getting the abosulte charge of colloids. On the basement of acid-base titration and Stat-pH titration, we tested the absolute charge of one point on△charge-pH curve and then moved them parallel using this point as reference. Finally we can obtain absolute charge of colloids as a function of pH at different KC1concentrations. For getting the absolute charge of HS, two methods were applied and compared. The first method was polyDADMAC titration and the absolute charge of HS was calculated according to the mass ratio polyDADMAC/HS at IEP of the complex and the charge of polyDADMAC (5.9mmol g-1); the second method was calculating the proton release by changing pH and ionic strength of protonated sample solutions. The results showed that the charge of JGHA obtained from the first method was larger than the second and for JGFA the first method was not suitable and made the result much lower.2. We monitored the change of potential (mV) and pH values when LSZ was titrated by HS. The mass ratio HS/LSZ at IEP of complex (mHS/mLSZ)IEP can be obtained with muitek particle charge detector. The results showed that the affinity of HS and LSZ was high and (mHS/mLSZ)IEP increased with increase of ionic strength and decrease of pH values. Moreover, we also calculated the mass ratio (mHS/mLSZ)CCP, which equals the charge density of LSZ divided by the charge density of HS. By comparison,(mHS/mLSZ)IEP>(mHS/mLSZ)CCP can be found, which is due to the participation of K+And the participation of K+increased with ionic strength and decreased with pH. Moreover, it was also related to the types of HS. The participation of K+reached the largest in JGFA-LSZ complexes and the lowest in PAHA-LSZ complexes.3. The flocculation of HS-LSZ complexes (with series of mass ratios) as a function of time was observed. The results showed that the aggregation of complex at IEP occurred the earliest and the amount of aggregation was the largest. For HA-LSZ complexes with larger mass ratio (f=1.0), the aggregation was inhibited. This was probably due to the electrostatic repulsion between negatively charged molecules.4. During HS interaction with LSZ, the enzyme activity in complexes was measured to reflect the effect of pH, ionic strength, mass ratio HS/enzyme on the formation, charge properties and structure of complexes, further indicating the mechanism of their interaction. Our results showed that electrostatic interaction plays an important role in the interaction of HS with LSZ. Driven by electrostatic attraction, LSZ was encapsulated into HS inner structure. As a result, the active sites of LSZ were shielded and substrate could not contact with them, leading the decrease of LSZ activity. Especially for two oppsitely charged particles, mass ratio was crucial because it determined the surface charge and structure of complexes; ionic strength screened the electrostatic interaction between HS and LSZ, which was also related to mass ratio. The effect of ionic strength on LSZ activity was opposite before and after IEP of the complex, indicating the importance of electrostatic interaction. Due to the smaller molecular weight and simpler structure of FA, the encapsulation of LSZ by FA was much weaker. The interaction of HS with amphoteric urease was also investigated. The electrostatic interaction was also important and was dependent on pH value of solution. When pH was larger than the PZC of urease, electrostatic repulsion hindered the encapsulation of urease by HS and maintained the high activity of urease; when pH was lower than the PZC of urease, electrostatic attraction improve the interaction of HS with urease, which was similar with LSZ-HS complex. The presence of HS decreased LSZ stability, and this inhibition reached maximum at the IEP of complexes due to the strongest aggregation of complexes at this point. However, the presence of HS improved the stability of urease. This was possibly because the collision among urease moleculars was decreased by HS and increased its stability.5. The fluorescence spectrum and synchronous excitation fluorescence spectrum (△λ,=60) of LSZ in the presence of HS at pH5and5mmol L-1KC1were conducted in order to investigate the change of microenvironment of LSZ. The results indicated that with excitation wave set at280nm, a progressive reduction in fluorescence intensity at340nm was caused by the increasing concentrations of HS. As mass ratio increased to0.2, the fluorescence intensity was nearly zero. Thus, the fluorescence of LSZ was strongly quenched and the red shift of λmax can be reasonably attributed to an increased polarity (or a decreased hydrophobicity) and loose structure of the region surrounding Trp site.6. The circular dichroism spectra of LSZ in the presence of HS were conducted and the secondary structure of LSZ in HS-LSZ complexes was calculated. Our results showed that the content of a-helix and random coils of HA-LSZ complexes firstly increased and then decreased with the increase of mass ratio HS/LSZ. For β-sheets, its content as a function of mass ratio HS/LSZ was opposite to a-helix and random coils. The turning points for the percentage of a-helix, random coils and β-sheets as a function of mass ratio HS/LSZ were formed at IEP of the complex, which was corresponding with the acticity of LSZ in complex as a function of mass ratio HS/LSZ. At this point, the complex of HS-LSZ started to disaggregate, accompanied with the change of secondard structure. For JGFA-LSZ complex, the content of a-helix, random coils and β-turns increased with increasing mass ratio while the content of β-sheets decreased.7. With ATR-FTIT spectrum, we investigated the effect of HS on the FTIR spectrum of LSZ at different conditions like incubation time, pH, ionic strength and mass ratio HS/LSZ. The results showed that the adsorption bands of LSZ were screened by HS most significantly around the IEP of HS-LSZ complex, Moreover, the mass ratio at which the screening was the most significant decreased with the increase of pH. This was corresponding to the decrease of (HS/LSZ)IEP with increasing pH. When ionic strength was increased to50mmol L-1, the similar results were not observed. As to JGFA-LSZ complex, factors like mass ratio, pH and ionic strength had little influence.