Studies On The Binding Of Heavy Metal Cd²⁺,zn²⁺ To Transferrin By Using Square Wave Voltammetry

Transferrin(Tf) is a serum iron transport protein. The Fe³⁺-Tf complexes transport Fe³⁺ across cell membranes by the transferrin receptor(TfR). Transferrin is a glycoprotein of approximately 80,000 kD, each of them consists of a single polypeptide chain of 680 amino acids, and 15 disulfide bonds, the C-terminus contains more disulfide bonds than N-terminal. The protein consists of two distinct lobes, each of them contains a high affinity metal binding site. The Tf-Fe is steadily at pH=7.4-10.0, release irons at pH=6.5, and released completely at pH=4.0. In the process of Tf combining with Fe³⁺, bicarbonate anion are needed, such as CO32-, HCO32-. They provide coordinate bond in the formation of Fe³⁺-Tf-CO32-, which made the binding steadily and prevent Fe³⁺ from combining with H2O. Fe³⁺ combining with one metal ions and release three H+ at the same time.Recently, with more and more heavy metal ions got into our environment and food chain, it is important to study the interactions between heavy metal ions and biomolecules. Normally the transferrin in serum is only 30% saturated with iron, so that the protein provides a significant concentration of vacant binding sites which can sequester other metal ions enter the blood. Then theTf-M could transport heavy metal ions into cells. Tf may be a mediater toxicological role in this process. Therefore, it is necessary to study the interactions between heavy metal ions and Tf.It is because the chemical properties of Cd, Zn are similar to Fe, so they can occupy the metal binding sites of Tf, and influence on series of biological functions. In this work, based on the highly sensitive polarographic signal of Cd²⁺,Zn²⁺, we have studied the capacity of Cd²⁺,Zn²⁺ bound to transferrin by using the square wave voltammetry. Our data show that Tf can sequester Cd²⁺,Zn²⁺. The main results appeared below.Fristly, the binding of Cd²⁺ to Tf was measured at pH=7.4 by using square wave voltammetry. Experimental results show that the concentration of the free Cd²⁺ in the Cd²⁺-Tf system is smaller than the total added concentration. It indicated that Cd²⁺ ion can enter into the metal binding sites in Tf. When adding different concentration of Cd²⁺ to Tf solution( CT f=10μmol/L), and CC d /CTf²⁺=0,0.2,0.4,…,6. Experimental results show that when CC d /CTf²⁺<0.8, the concentration of free Cd²⁺ in the Cd²⁺-Tf system is nearly nil. It means that Tf nearly bound Cd²⁺ quantitatively in this concentration range, with high speed, strong force and Cd²⁺ into the strong binding sites in Tf. When C Cd /CTf²⁺>0.8, the concentration of free Cd²⁺ increase gradually in the Cd²⁺-Tf system, and n (the number of Cd²⁺ bound per Tf ) also get bigger gradually. It showed that the binding force between Cd²⁺ and Tf becomes weaker than that in the first phase. By fitting the binding curves of Cd²⁺ to Tf by using simplex algorithm, the calculated binding constants of the two sitess are lgK1=6.57, lgK2=3.73. The results showed that the two metallic ion combining sites of Tf could combined with Cd²⁺, but unsaturated.Secondly, the binding of Zn²⁺ to Tf was measured at pH=7.4 by using square wave voltammetry. The results showed that the concentration of the free Zn²⁺ in the Zn²⁺-Tf system is smaller than the added total concentration. In Tf solution( CT f=1.00×10-5mol/L), we added Zn²⁺ ions gradually, and C Zn /CTf²⁺= 0,0.2,0.4,…,20. Experimental results show that when /≤C Zn 2 +CTf1, the concentration of free Zn²⁺ in the Zn²⁺-Tf system is nearly nil, it indicated that the binding force betweenTf and Zn²⁺ is strong and there is a strong Zn²⁺ binging site in Tf. When C Zn /CTf²⁺>1, the concentration of free Zn²⁺ increase gradually. But the reduction peaks of Zn²⁺ (Ep) in Zn²⁺-Tf is lower about 30mV than that in Zn²⁺ solution, and the peak current of Zn²⁺ (Ip) is also lower than that in Zn²⁺ solution. It showed that Tf has a dramatic impact on the double electrode layer structure of the mercury electrode and the redox reaction rate of Zn²⁺. So we can not get the accurate concentration of free Zn²⁺ in the Zn²⁺-Tf system by Zn²⁺calibration curve and the more details about Tf bound Zn²⁺. Other methods to describe the character of the binding of Zn²⁺ to Tf are needed.Thirdly the binding of Pb²⁺ to Tf was measured at pH=7.4 by using square wave voltammetry,and no signal of Pb²⁺ were detected in Tf-Pb²⁺ system.In addition, we also studied whether add NaHCO3 to buffer solution has influence on the binding of metal ions to Tf .