Study On The Interaction Of Persimmon Tannin With Bovine Serum Albumin And Phospholipase A₂

Serum albumins are the major soluble protein constituent in the circulatory system, which play an important role in the transportation and deposition of many drug molecules in the blood. It is known that the distribution, free concentration and the metabolism of various drugs can be significantly altered as a result of their binding to serum albumin. Therefore, investigating the interaction of drugs and serum albumins is significant for knowing the transportation and distribution of drugs in body. As we known,the composition of venom is very complicated,containing a variety of toxins、enzymes、proteins、peptides, etc. And its pathological effects such as neurological toxicity, muscle toxicity, bleeding toxicity and lethality are closely related to proteases contained in Snake venom. PLA2 exerts many pharmacological activities including neurotoxicity, myotoxicity, cardiotoxicity, anticoagulant and antiplatelet. Our previous study suggested that persimmon tannin has significant inhibition on snake venoms.To understand the detoxifying mechanism of persimmon tannin on snake venoms, this experiment studied the binding of persimmon tannin to BSA and PLA2 under different conditions.The fluorescence spectroscopy combined with UV-visible absorption spectroscopy, Fourier transform infrared(FT-IR) spectroscopy、circular dichroism (CD) and SDS-PAGE analysis were used to investigate the interaction of persimmon tannin with BSA and PLA2.The main results were shown as follow:1. PT40 was found to have stronger aggregation ability with BSA than HMWT.The precipitation rate reached maximum when the ratio of BSA to PT40 reached 5.75:1.The feasible aggregation between PT40 and BSA was observed at 30℃under pH 4.0, time had no significant effect on the binding. The interaction of PT40 with BSA was studied by fluorescence spectroscopy under simulative physiological conditions.And the Stern-Volmer plots and the Scatchard plots were applied to analyze the data.The Stern-Volmer plots has two regression curves intersecting at [PT40]≈2.06μM with [PT40] concentrations 0-2.06μM and 2.06μM～4.05μM,and the plots deflected to Y axes.The Scatchard plots also has two regression curves, indicating the binding of PT40 to BSA with two types of sites.When [PT40]<2.06μM, the numbers of binding sites were near 1.4, and when 2.06μM<[PT40]<4.05μM,the numbers were approximately 2.0.The FT-IR and CD spetra proved that the secondary structure of BSA changed after interacting with PT40.Thermodynamic analysis showed that hydrophobic interactions was the mainly binding force and it’s a spontaneity、exothermic reaction.Resonance light scattering analysis shows that it didn’t formed clear aggregates between BSA and PT40.2. PT40 was found to have stronger aggregation ability with PLA2 than HMWT.The precipitation rate reached maximum when the ratio of PLA2 to PT40 reached 2.48:1. The greatest aggregation between PT40 and BSA was seen at 40℃under pH 4.5-5.0., and time had no significant effect on the binding.The Stern-Volmer plots has two regression curves intersecting at [PT40]≈0.78μM with [PT40] concentrations 0～0.78μM and 0.78μM～2.025μM,and the plots deflected to Y axes.The Scatchard plots also has two regression curves,suggesting the binding of PT40 to PLA2 with two types of sites.When [PT40]<0.78μM, the numbers of binding sites were near 1.2, and when 0.78μM<[PT40]<2.025μM,the numbers were approximately 1.9.The FT-IR and CD analyses proved that the secondary structure of PLA2 changed significantly after interacting with PT40.Thermodynamic analysis showed that hydrophobic interactions was the mainly binding force.Resonance light scattering analysis shows that it formed clear aggregates between PLA2 and PT40.3. The competitive binding of BSA and PLA2 to PT40 was studied by blue BSA method.The results showed that, PT40 have stronger aggregation ability with BSA than with PLA2 at pH 4.9; while, at pH 7.4, PT40 showed stronger affinity with PLA2 than with BSA. In addition, in a competitive condition, PLA2 could capture PT40 that have been binded by BSA, suggesting the complex of PLA2 and PT40 was more stable than the complex between PT40 and BSA.4. The His residues and Lys residues from PLA2 were modified by p-BPB、acetic anhydride, separately. It was found that the modification resulted in a strong inhibition of enzyme activities, as well as a notable decreases in the hemolysis and edema-inducing activity of PLA2. Furthermore, the lethal toxicity of PLA2 decreased significantly after chemical modification. Acute toxicity test on PLA2 showed that it’s LD50 for mice (i.p) was 5.645mg/kg. After reaction with PT40, the LD50 increased to 11.061mg/kg. Treating PLA2 with PT40 also resulted in a remarkable changes on the catalytic activity, hemolysis and edema-inducing activity of PLA2. SDS-PAGE combining western-blot and NBT stain analysis showed that PT40 might inhibit the activity and toxicity through covalent modification of the Lysine residues, which is very important for PLA2’s catalytic activity and pharmacological activity.