Interactions Of Adriamycin With Human Serum Albumin And Cardiac Myosin

Adriamycin (Doxorubicin, ADR) separated from Streptomyces peucetius was a anthracycline-based antibiotic. It was one of the importance drugs in the field of chemotherapeutic and widely used in treatment of various human tumour diseases. The major amount of ADR in the blood stream was most likely bound to a well-known transport protein - Human serum albumin(HSA). With blood circulating round the body, ADR had a considerable impact on various organs. The clinical application of ADR was limited by its toxic dose - related side effects, especially such as myelosuppression and cumulative cardiotoxicity. In this paper, in an attempt to obtain some detailed experimented and theoretical information on the transport of ADR in human body and the mechanism of the serious cardiotoxicity of ADR, the interactions of ADR with HSA and cardiac myosin were studied.1. The binding of ADR and its iron complex with HSA was studied by the resonant mirror biosensor (IAsys). The results showed that the binding reaction was enhanced with temperature rising, and the affinity of ADR-Fe(III) with HSA was higher than that of ADR. The binding reaction was driven largely by the entropy change. Therefore, the binding process was governed primarily by hydrophobic force, and the water molecules played a major role in the binding reaction.2. The interaction of ADR and HSA was studied by fluorescence spectroscopy. First, the effect of pH on the binding of ADR and HSA was presented. The results showed that ADR has an ability to quench HSA fluorescence and compared with under other pH conditions, the Stern-Volmer quenching constants was lower and the binding distance was larger under pH 4.00. Non-radiative energy transfer was the reason of fluorescence quenching. It was not obviously observed influence on the binding of ADR and HSA under neutral pH, subacidity and alkalescent conditions, electrostatic interaction was not the main binding force. Based on the binding distances of Trp~214 residue to the binding site of ADR, we presumed that various fatty acids would affect the function and distribution of ADR in human body. Second, the effect of different buffer on the interaction of ADR and HSA was studied. The results showed that the Stern-Volmer constant tread with pH wasn't influenced by different buffer, and Na2HPO4-KH2PO4 buffer was helpful to the binding of ADR and HSA. Third, under pH 4.97 and pH 7.41, the effect of temperature on the interaction of ADR and HSA was studied. The results indicated that during 20 °C to 30°C, the fluorescence quenching of HSA was static quenching term, probably, ADR reacted with HSA, and ground state compound appeared; but during 15°C to 37 °C, the fluorescence quenching of HSA was collisional quenching term, HSA fluorescence quenched due to the transfer of energy or electron between ADR and HSA. The phenomena were obvious under pH 7.41. The fluorescence quenching of HSA included static quenching and collisional quenching term, the mechanism of the interaction of ADR and HSA was complex. Forth, the effect of ionic strength on the interaction of ADR and HSA was studied. The results showed that the effect of ionic strength was neglected in the interaction of ADR and HSA, it indicated again that electrostatic interaction forces wasn't the major contributors to the binding of ADR with HSA.In this work, HSA fluorescence quenched by ADR, but the HSA fluorescence spectroscopy treads with pH, temperature and ionic strength weren't affected by ADR. It indicated that ADR acted with HSA, but the force wasn't strong. Hydrophobic and Van der Waals forces were the major contributors to the binding interaction. The binding process wasn't governed by electrostatic interactions. HSA is a storage andtransport protein for ADR.3. After His and Cys were modified, the effect of ADR with modified HSA was studied. The results showed that the fluorescence quenching term was collisional quenching term after His was modified, and the interaction was decreased, so His residues were essential for the interaction of ADR and HSA. The fluorescence quenching term was static quenching term after Cys was modified, and ground state compound appeared. The binding constants of ADR with modified HSA obtained from Lineweaver-Burk equation reflected that the interaction mechanism was complex.4. Autodock program was used to calculate the interaction modes between ADR and HSA. The results showed that the four lowest docked energy sites were similar with some binding sites of fatty acids with HSA, so various fatty acids would affect the binding of ADR with HSA. The amino acid residues around 4 A of ADR of the four lowest docked energy sites were studied, lots of hydrophobic amino acids were essential for the stability of the binding interaction. His played a major role in the binding of ADR with HSA. The results calculated through Autodock were nearly in accordance with the experiment results.5. Based on the solubility of cardiac myosin was notable diversity under different ionic strength solution. Myosin was separated, then purified by saturation (NH^SC^, the fraction between 36% and 41% saturation with (NH^SC^ was collected. Pure myosin was detected by SDS—PAGE; The ATPase activity of the purified myosin was measured by an improved and sensitive colorimetric assay.6. The interaction of ADR with home-made pure myosin was studied. The results showed that with higher of the concentration and longer of the reaction time, the lose of myosin ATPase activity was more serious, under the condition of 10 mmolL"1 Ca2+, the Stern-Volmer constant of the interaction of ADR with myosin was obviously higher than that under the condition of 5 mmolL"1 Ca2+. Under some concentration range, with the increase of the concentration of Ca2+, the interaction of ADR and myosin was enhanced, Ca2+ played a major role in the binding reaction.