Purification Of Cardiac Myosin And Its Fluorescence Studies Interacted With Daunorubicin

Daunorubicin (DNR) is one of the most important anthracyclines, whose anticancer activity is linked with DNA and inhibition of both DNA and RNA synthesis. However, if the patients take it for a long-term, they will suffer from cardiotoxic symptoms and the definite mechanism is still disagreed. Nevertheless, it was reported that DNR may contribute to destruction of actin–myosin cross-bridge machinery of the contractile cardiac muscle. Cardiac myosin (CM) is primary structure protein and contractile protein in heart muscle cells, whose ATPase activity exhibits the function status of the cardiac cells and influence the contractile capacity. It is a foundation that the article focuses on the interaction of DNR with CM. Moreover, because of its electron donator, DNR can form co-ordinate complex with protein. It was known that the proteins interacted with DNR concentrate on bovine serum albumin (BSA) and human serum albumin (HSA), which CM almost hasn't been reported. The purpose and implication of the research is to investigate the binding mechanism of DNR and CM, demonstrate the reasons of changes and cardiac toxicity carried by DNR, as well as provide some reasonable explanations about cardiac toxicity in molecular level from the variations of ATPase and fluorescence spectrum of CM.Several latter-day methods about reacting pharmaceutical and protein are narrated in this paper, including sensitive and convenient fluorospectrophotometry. General approaches and depuratus results are summarized and improved before CM is extracted from porcine left ventricle. In addition, the effect of DNR on ATPase activity and fluorescence quenching pattern of CM are discussed. The major contents in the study are described as follows:1. Purification CM and determination ATPase activityCM is rarefied from porcine left ventricle by using of 36%～41% saturation (NH₄)₂SO₄. The contents of CM in every separating process are measured and corresponding Ca²⁺-ATPase activities are determined, based on the ATP quantity released by CM, in addition to graphing ATPase kinetics curve. The CM activity recovery and purification multiple is 39.4% and 4.7, respectively. The decontamination degree is identified by SDS-PAGE and HPLC. Compared with traditional approaches, the method is simple and quick operation, high recovery and ATPase activity.2. Effect of DNR on CM Ca²⁺-ATPase activityDetermining protein activity is an important part in purifying process and Ca²⁺-ATPase activity is one of the most effective indicator to CM. So, the influence of DNR on CM Ca²⁺-ATPase activity is studied. The result shows that Ca²⁺-ATPase activity decreases until effect CM normal physiological functions with the increase of DNR in dose and time.The effect of Mg²⁺ concentration on Ca²⁺-ATPase activity of CM and DNR-CM system is compared. It enunciates that DNR-CM Ca²⁺-ATPase activity is smaller than CM system in same Mg²⁺ concentration, and the loss of Ca²⁺-ATPase activity is controlled by trace Mg²⁺.The influence of pH on ATPase activity of DNR-CM system is also investigated. It exhibits that Mg ²⁺-ATPase activity is greater than Ca²⁺-ATPase activity in same pH value and the higher Ca²⁺-ATPase activity is one of the reasons that make cancer patients heart-failure in acid environment.3. DNR and CM fluorescence actionThe effect of DNR on CM fluorescence spectrum in pH7.4 Tris-HCl buffer and at 20℃is investigated.The result shows that CM Stern-Volmer curve is not absolute linear, therefore, the quenching of DNR and CM is not dynamic but static. The quenching constant and binding constant is 1.337×10⁵ L·mol^-1 and 2.649×10² L·mol^-1, respectively. The binding of DNR to CM is a spontaneous molecular process, coupling with the reduction of free energy.The effect of pH, buffer system and temperature on DNR and CM fluorescence quenching is also studied. The quenching constant is greatly affected by pH and gradual increased according to the sequence: pH6.5⁷.4(neutrality), pH8.0⁹.0(weak base) and pH4.0⁶.0(weak acid). Moreover, the binding constant in pH5.5 is highest than other pH.The system quenching degree is strongest in Tris-HCl buffer than that in Citric acid-Na₂HPO₄ and KH₂PO4-K₂HPO₄ buffers and the quenching constant keeps invariable in pH6.5⁷.4 Tris-HCl buffer.CM fluorescence quenching style is deduced by quenching constants in 15℃³7℃. In addition, the binding of DNR to CM is firmest in physiological temperature. 4. Effect of metal ions on DNR and CM fluorescence quenchingNot only CM fluorescence intensity can be quenched, but also the quenching action between DNR and CM be strengthened by Ca²⁺ and Mg²⁺. The system quenching constants and binding constants are obtained in different ionic concentration. The causes about DNR cardiac toxicity in abnormal concentration are provided.Compared with Cu²⁺, the quenching mechanism of Zn²⁺and CM is complicated, which embraces dynamic and static process simultaneously. Metal ions inhibit the binding of DNR to CM, decreasing the quenching reaction and restraining DNR cardiac toxicity, as Zn²⁺and Cu²⁺ concentration rate is 10 to 1. When the rate is 100 to 1, the quenching constant is maximum, which shows that the binding strength is stronger. The result is in agreement with the information that Zn²⁺and Cu²⁺ concentration rate is higher in cancer patients.CM fluorescence intensity is decreased monotonously andâ–³F is smaller and smaller with increasing Se⁴⁺ concentration. However, as for DNR-CM system, fluorescence intensity shows three different tendencies, such as quenching, sensibilization and invariability. When Se⁴⁺concentration is 1.2×10^-5 mol·L^-1, a new fluorescence method is established to determine DNR, based on the fine linear relationship betweenâ–³F and DNR concentration. When Se4+concentration is 2.0×10^-5 mol·L^-1, the fluorescence intensity keeps constant with increasing DNR, which illustrates that Se⁴⁺is helpful to protect heart from DNR side effect.