Electrochemical Studies On Interactions Of Baicalein, Entecavir And Tamoxifen With Protein And DNA

The study on the interaction of large biological molecules with small drug molecules was the popular research topics in life science.Among the methods employed for studying the interaction of biological molecules with small molecules,electrochemical method possessed the advantages such as high sensitivity,rapid response,simplicity in operation procedure,and could obtain information about interaction mechanism.In this paper,baicalein,entecavir and tamoxifen were chosen as small molecule research subjects,voltammetry and zero current potentiometry were employed for studying the interactions between the drugs with DNA and protein.The details were given as follows:1 The electrochemical oxidation of antioxidant baicalein(BA) and its interaction with human serum albumin(HSA) were investigated in Britton-Robinson buffer(pH 3.3) in the present work.The results showed that BA was oxidized to its o-diquinone derivative through a two-electron transfer of 6,7-dihydroxyl groups on A-ring via an intermediate free radical at lower potential 0.35 V.Then a portion of the formed o-diquinone derivative was further oxidized to its quinone derivative through a two-electron transfer of 5-hydroxyl group at higher potential 0.71 V,and the other o-diquinone derivative was reduced to BA at lower potential 0.32 V.In addition,BA can combine with HSA rapidly,forming a stable electro-inactive complex(HSA·BA₂) with binding ratio BA:HSA=2:1 and apparent binding constantβ=(3.26±0.15)×10⁹(mol·L^-1)^-2(P=0.90).2 The interaction between BA and calf thymus dsDNA(DNA) was investigated at carbon paste electrode(CPE) and DNA modified carbon paste electrode(DNA/CPE) by cyclic voltammetry and differential pulse voltammetry.In BR(pH 3.3) buffer,the oxidation peak potential of BA shifted in positive direction,and the peak current of BA decreased when it reacted with DNA in solution and on the DNA/CPE surface.These results demonstrated that BA combined with DNA through intercalative interaction and formed a complex DNA·BA, with the binding ratio 1:1 and the apparent binding constantβ6.07×10⁷(mol·L^-1)^-1.In addition, the oxidation peak currents of both guanine and adenine at DNA/CPE increased with the increase of BA concentration in BR(pH 3.3) buffer,which was ascribed to the damage of DNA double stranded helix structure by both BA and its oxidation product intermediate free radical.Moreover,the relationship of the oxidation peak current of guanine at DNA/CPE with BA concentration in solution showed a titration curve that BA titrating DNA.The curve can provide some information about the surface layer quantity of DNA immobilized on CPE.3 The interaction between entecavir(ETV) and bovine serum albumin(BSA) was investigated by single-sweep voltammetry at carbon paste electrode.In HAc-NaAc(pH 4.4) buffer,ETV presented an oxidation peak at 1.1 V(vs.SCE).After the addition of BSA into the solution containing ETV,the peak current of ETV was decreased obviously and the peak potential of ETV was nearly unchanged.The experimental results showed that ETV interacted with BSA and formed an electro-inactive complex.According to the different value of the ETV oxidation peak current with and without BSA,the binding ratio(m:1) of ETV and BSA was calculated to be 2:1 and the apparent binding constantβwas 2.37×10⁴(mol·L^-1)^-2.4 A new electrochemical method named as zero current potentiometry for studying the interaction between DNA and small molecule was proposed and the relational formulas for calculating the thermodynamic and dynamic constants of the binding reaction were deduced. In BR(pH 3.3) buffer,the interaction between DNA and BA was investigated at DNA/CP by zero current potentiometry.The thermodynamic and dynamic constants of the binding reaction were determined based on the shift of zero current potential of DNA/CP in BR(pH 3.3) buffer containing BA caused by the interaction between DNA and BA.The binding ratio m of the formed complex DNA·BA_m was calculated to be 1:1,and apparent binding constantβwas 7.58×10⁷ L·mol^-1.The apparent rate constant k_a of the binding reaction was 2.33×10³ (mol·L^-1)^-1·s^-1.In addition,the interaction between the electrochemical damaged DNA(DNA_ox) and BA was studied by the same procedure.The binding ratio m of the formed complex DNA_ox·BA_m was calculated to be 1:1,and apparent binding constantβwas 4.92×10¹⁰ L·mol^-1. The apparent rate constant k_a of the binding reaction was 4.29×10³(mol·L^-1)^-1·s^-1.5 In PBS(pH 7.0) buffer,the voltammetric behaviors of both tamoxifen(TAM) and DNA were investigated.The result showed that the oxidation peaks of TAM and DNA were overlapped each other.It was difficult to study the interaction between them by voltammetry. Immobilized DNA on the carbon paste(CP) surface,it was found that the zero current potential of DNA/CP in PBS(pH 7.0) buffer was shifted with the increase of TAM concentration.On the basis of the shift of zero current potential,the interaction between TAM and DNA was studied by zero current potentiometry.The binding ratio of DNA with TAM was calculated to be 1:1 and the apparent binding constant was 2.55×10⁸ L·mol^-1.