Applications Of Resonance Rayleigh Scattering Technique In Determination Of Bleomycins Antitumor Drugs

Resonance Rayleigh scattering (RRS) is a new analytical technique developed in 1990s. It has high sensitivity and good selectivity in studying the aggregation of chromophores on biological macromolecules and the ion-association between the anions and cations, and in studying the nonbonding action of biological macromolecules, in particular. Because of these merits, RRS has been used as a useful method for the study and determination of biological macromolecules, and this new technique has showed great advantages in the researches of nucleic acids.As the development of molecular biology, people pay extensive attentions to study the modes of interaction of small molecules with biological macromolecules at the molecular level, in which studying on the interaction of DNA with anticancer drugs has been a more active field. The study not only can elucidate the action mechanisms of some antitumor drugs, but also can provide useful information for designing the new-type anticancer drugs. In this dissertation, RRS technique was used to study the interaction of bleomycins antitumors with biological macromolecules such as nucleic acids and proteins. RRS methods were established to the determination of these biological macromolecules by using bleomycins as probes. In addition, the applications of RRS technique in the determination of bleomycins antitumors were developed.The main contents and some conclusions of the dissertation are as follows:1 Resonance Rayleigh scattering study of the interactions of bleomycin and metallo-bleomycin with nucleic acids and their analytical application(1) Resonance Rayleigh scattering study of the interaction of bleomycinA₅ with nucleic acids and its analytical applicationThe interaction of bleomycinA₅ with nucleic acids has been investigated by using resonance Rayleigh scattering (RRS), molecular absorption and fluorescence spectra. The results show that in near pH 2.2 buffer medium, nucleic acids are capable of binding with bleomycinA₅ (BLMA₅) to form complexes which can remarkably enhance the RRS intensity and result in bathochromic and hyperchromic molecular absorption of nucleic acids and fluorescence quenching of bleomycinA₅. The RRS spectral characteristics for the binding products of bleomycinA₅ with various DNA and RNA are similar and the maximum RRS peaks are at 301nm for ctDNA and sDNA, 370nm for hsDNA, 310nm for RNAtypeVI and RNAtypeIII, respectively. The increments of RRS intensity are great different in which DNA enhances greatly and RNA enhances lightly. In this work, the optimum conditions of the interaction and some influencing factors have been investigated. The reaction mechanism and a binding model for the interaction of BLMA₅ with the nucleic acids are discussed. In addition, a highly sensitive, simple and rapid new method for the determination of DNA has been developed. The detection limits (3a) are 5.7ng mL^-1 for ctDNA, 7.4ng mL^-1 for sDNA and 9.2 ng mL^-1 for hsDNA, respectively. The method was applied to determination of DNA in synthetic samples and practical samples with satisfactory results.(2) Study on the interaction of Cu(II)-Bleomycin A₂ and Fe(II)-BleomycinA₂ with nucleic acids by resonance Rayleigh scattering, UV-spectroscopy and fluorescence method and their analytical applicationUsing nucleic acids as analytes, the RRS spectra, the fluorescence spectra and the absorption spectra of the interaction of bleomycinA2(BLMA2) with copper ion Cu(II) and ferrous ion Fe(II) and their analytical application have been studied. The results show that in certain acidic medium, BLMA2 can react with Cu(II) and Fe(II) to form cation chelates which result in the changes of the absorption spectra and the quenched fluorescence of bleomycinA2. When the cation chelates further react with different nucleic acid, the great enhancements of RRS intensities are observed. Their RRS spectral characteristics are similar, and the maximum RRS wavelengths are at 301 nm. The increments of RRS intensity are different in the series of Cu (II)>Fe(II). In this work, the effects of acidity on the formation of chelates and the interaction with nucleic acids have been investigated, further more, the changes of UV-spectra, fluorescence spectra and RRS of the reaction systems are discussed. In addition, using Cu(II)-BLMA₂ as an example, a highly sensitive, simple and rapid new method is proposed to the determination of DNA. The detection limits (3a) are 7.2ng mL^-1, 7.05ng mL^-1 and 18.3 ng mL^-1 for ctDNA, sDNA and hsDNA, respectively. 2 Resonance Rayleigh scattering, absorption spectra and fluorescence spectra of bleomycins- halofluorescein systems and their analytical applicationThe interaction of bleomycins with halofluorescein dyes such as erythrosin (Ery), eosin Y (EY), eosin B (EB) and rose bengal (RB) has been studied using the RRS, UV-spectroscopy and fluorescence quenching technology. The results show that the binding of bleomycins with halofluorescein dyes can result in a significant enhancement of RRS, the fading reactions and the fluorescence quenched of four halofluorescein dyes. The decrements of absorbance (ΔA), the fluorescence quenching values (ΔF) and the increments of scattered intensity (ΔI) are directly proportional to the concentrations of bleomycins in a certain range. The new three methods for the determination of bleomycins based on the fading reactions, fluorescence quenched and RRS have been developed. The methods exhibit higher sensitivity than that of common spectrophotometry and fluorescence methods. Among them, RRS has the highest sensitivity. The detection limits (3a) for BLMA5 and BLMA2 are 16.6 ng mL^-1 and 18.0ng mL^-1, respectively. In this work, the optimum conditions of the reaction are investigated. By combining the quantum chemistry calculation, the formation of ion-association complexes and the effects on the absorption spectra and the fluorescence, as well as the reasons of enhanced resonance light scattering are discussed.3 Resonance Rayleigh scattering studies of the interaction of metallo-bleomycin with proteins and some small dyes molecules and their analytical application(1) Study on the interaction of Cu(II)-BLMA₅ with protein by resonance Rayleigh scattering and its analytical application.Under optimum conditions, Cu (II) -BLMA5 complex aggregates on the surface of the protein to form a new super-molecular complex. The molecular weight enhancement of scattering particles and the hydrophobicity of the super-molecular complex enhance the RRS signal. The maximum RRS peaks for bovine serum albumin (BSA) and human serum albumin (HSA) are all at 550nm. The enhanced RRS intensity is proportional to the concentration of proteins in the range of 0.098～15μgmL^-1 for BSA and 0.16～15μg mL^-1 for HSA. The detection limits (3σ) are 0.028μgmL^-1 for BSA and 0.048μgmL^-1 for HSA. The results of the determination of serum samples are close to those obtained from the Coomassie Brilliant Blue method.(2) Study on the interaction of metallobleomycins with Ery and its analysis application by resonance Rayleigh scattering methodIn weakly acidic buffer medium (pH4.0), the bleomycinA₅ reacts with Cu(II) and Co(II) ion to form 1:1 cation chelates which further react with anion dye Ery to form ternary association-complexes. As a result, the significant enhancements of RRS intensities are observed. Their spectral characteristics of RRS are similar, and the maximum RRS wavelengths are at 287 nm. The increments of RRS intensity are different in the series of Co (II) > Cu (II). The enhanced RRS intensities are proportional to the concentration of BLMA₅ in the range of 0.048～4.8μgmL^-1 for Co(II) system and 0.066～8.0μgmL^-1 for Cu(II) system. The detection limits (3a) are 14.4 ngmL^-1 for Co( II )system andl9.8 ngmL^-1 for Cu(II) system. Compared with the binary Ery-BLMA systems, the ternary systems have wider linear ranges for determination.(3) Resonance Rayleigh scattering studies of the interaction of Ce(IV) -BLMA₅ with Congo Red(CR) and its analytical application.The combination reaction of BLMA₅-Ce (IV) with CR has been studied using RRS technology. The results show that in the HAc-NaAc buffer solution (pH 5.4), BLMA5 can react with Ce(IV)to form cation chelates which further react with CR by virtue of electrostatic attraction and hydrophobic force to form ion-association complex. As a result, the resonance Rayleigh scattering (RRS) enhanced greatly. A new method for the determination of bleomycins based on RRS has been developed. The enhanced RRS intensity is proportional to the concentration of BLMA5 in the range of 0.51～10μgmL^-1 and the detection limits (3σ) is 0.15μgmL^-1 for BLMA5. This method provides a new mean to study of the interaction of rare earth ions with bleomycins.