Study On The Binding Mode And Capacity Of Aflatoxin B₁ To Free DNA

In order to study the binding mode and capacity of aflatoxin B₁?AFB₁?to free herring sperm DNA?DNA?,the interaction mechanism between AFB₁ and DNA was studied through various analytical methods.The DNA binding saturation value?DNA BSV?of aflatoxin B₁ to DNA was calculated by resonance light scattering?RLS?method.The effects of various factors such as pH,temperature,ionic strength and other environmental coexistences on the behavior of aflatoxin B₁ to AFB₁ to free DNA were also studied.The interaction mechanism of AFB₁ to DNA in vitro was studied.The results of DNA thermal denaturation temperature?Tm?showed that the Tm of DNA increased by about 13.1 C,which may be due to the denser and more stable DNA double helix,and the intercalation of AFB₁ between the base pairs of DNA.Ultraviolet-visible spectra showed that the structure of DNA has been changed and a new AFB₁-DNA complex has been formed,and the interacting mode was intercalation.Fluorescence quenching spectra also showed that there was a competitive interaction between AFB₁ and ethidium bromide?EB?to form AFB1-DNA complex.In addition,the enhancement of negative peak signal at 245 nm and the change of positive peak signal at 270 nm in Circular Dichroism spectra indicated that DNA base-pair stacking has changed,and the content of B-DNA has increased.And there is a significant increased dose-dependent negative peak near 275 nm,suggesting that AFB₁ may be a DNA intercalator.The results of agarose gel electrophoresis showed that DNA move to the positive electrode which carried AFB₁ molecules and emited blue in the lane,indicating that their binding mode was intercalation.All these results indicated that the interaction mode between DNA and AFB₁ is an intercalating mode in vitro.Aflatoxin B₁ can interact spontaneously with DNA in vitro and its interacting mode is intercalation,which also provides theoretical support for the elimination of aflatoxin by DNA selectively binding to aflatoxin and separation of aflatoxin-DNA system by oil-water separation or magnetic bead adsorption.Based on the analysis of fluorescence,the results of double logarithmic regression curves showed that the binding constant of AFB₁competitivelybinding to EB-DNA was 5.58L/mol,and the binding site of EB-DNA molecule was substituted by about 1 AFB₁ molecule,and the binding constant was weak.The binding constant of AFB₁ to DNA was obtained through the method of Scatchard equation,which also proved that the interacting mode belonged to intercalation and they are basically consistent with those obtained by ultraviolet titration.The thermodynamic data showed that the formation of AFB1-DNA complexes was a spontaneous binding reaction without any enzymatic oxidation andthe main driving forces were Van der Waals force and hydrogen bonding.Resonance light scattering spectroscopy showed that the DNA BSV of AFB₁,ethidium bromide,psoralen and angelicin binding to DNA were 15.59,2.14,0.74 and 0.74,respectively.The binding capacity of AFB₁ to DNA is weaker than that of EB,but stronger than those of PSO and ANG.The study on binding capacity of AFB₁ to DNA in vitro provides quantitative reference for eliminating aflatoxin by selective binding to DNA.The factors affecting the interaction of AFB₁ to DNA were studied by resonance scattering spectroscopy.The optimum conditions for the formation of AFB₁-DNA system were pH 7.40,30 C,and lower ionic strength,and DNA BSV was 3.17.The conditions for strong acid and strong base are not conducive to the formation of system.Temperature and cations are sensitive to the RLS signal intensity.Glucose,nicotinic acid and aminoacetic acid can enhance the RLS signal intensity of AFB₁-DNA system,but they can reduce the DNA BSV value.However,L-lysine,glycerol,urea,glucan and composite protease will have a greater impact on the binding reaction between AFB₁ and DNA.Therefore,the presence of other environmental coexistences should be minimized as possible in the determination process.The study on factors affecting the binding capacity of aflatoxin B₁ to DNA can provide and establish more suitable conditions for the formation of aflatoxin-DNA system and the separation of their complex.