【作者】 王彦卿；

【导师】 郭子建；

【作者基本信息】 南京大学 ， 无机化学， 2011， 博士

【摘要】 蛋白质是生命体系中最重要的一类生物大分子,有关药物分子与蛋白质相互作用的研究一直是分子生物学中最重要的领域之一。人血清白蛋白(HSA)是人体血浆中含量最丰富的蛋白质,具有转运药物分子的功能。人血红蛋白(Hb)是人体内执行输氧任务的蛋白质,γ-球蛋白(γ-Gb)是最主要的免疫球蛋白之一,亦能够结合药物分子和相关的抗原。为了降低顺铂的毒副作用和克服其抗药性,许多非经典的具有新型结构的铂类配合物不断的被设计出来。关于具有抗癌活性的新型铂类配合物与蛋白质的相互作用对于评价金属配合物的药理及潜在的毒性有着重要意义,同时在指导新型金属药物设计、金属药物药代动力学研究等也具有指导性意义。本研究在总结了目前研究金属配合物与蛋白质相互作用的方法及原理后,选用本课题组设计合成的新型单功能铂类配合物,8-氨基喹啉铂(8-QA-Pt)、萘酰亚胺铂配合物(PEN-Pt)、4,4’-二胺二苯基甲烷为配体的双核铂类配合物(Pt2-L)及以三齿螯合的多吡啶为配体的三核铂配合物(Pt3-L)作为模型药物,利用荧光、紫外、CD、FT-IR、MALDI-TOF及分子模拟等技术研究了该类铂配合物与HSA.Hb及γ-Gb的相互作用,结果发现,该类配合物与蛋白质的作用方式明显不同于顺铂,8-QA-Pt、PEN-Pt及Pt3-L与HSA、Hb及γ-Gb主要以非共价键结合为主,而Pt2-L与HSA存在着共价键结合,与Hb及γ-Gb主要以非共价键结合为主；通过荧光猝灭方法得到了铂类配合物与蛋白质分子间的结合常数和热力学参数,利用作用位点竞争、蛋白质变性及分子模拟等方法研究表明,8-QA-Pt结合在HSA的Subdomain IB内,其它几种铂配合物主要与Subdomain IIA内的氨基酸发生作用,该类铂配合物在蛋白质分子上主要存在着一类结合位点,另外通过分子对接的方式确定了铂类配合物在Hb及γ-Gb的结合区域和结合模式；并用同步荧光、三维荧光、紫外光谱、CD、FT-IR等技术考察了铂配合物对蛋白质微结构的影响,结果发现该类铂配合物未引起蛋白质结构的明显改变。该研究内容有助于解释不同于顺铂的非经典铂类配合物的药理及药代动力学以及指导设计新型的铂类抗癌药物,通过选择合适的配体形成新型的铂配合物,实现不同于顺铂等经典铂类药物与常见转运蛋白的结合方式,提高有效药物的浓度和发挥药效的新方式。对蛋白质进行选择性的定性、定量的检测在生命科学、医药、和生物化学等方面具有重要意义。利用荧光探针对生物大分子进行定性、定量的选择性检测是许多生物化学家所关心的热点问题,关于利用荧光探针选择性识别乙醇脱氢酶的报道较少。本研究中选用可见光激发的7-硝基苯并呋咱(NBD)类荧光团新型的锌离子探针NBD-TPEA作为识别蛋白质的研究对象,研究其对乙醇脱氢酶(ADH)、碳酸酐酶(CAH)、谷氨酸脱氢酶(GDH)、牛血清白蛋白(BSA)、人血清白蛋白(HSA)、溶菌酶(Lys)、胰蛋白酶(Trypsin、γ-球蛋白(γ-Gb)等不同结构和功能的生物大分子的荧光响应性质,从而说明NBD-TPEA探针的生物学性质。此外重点研究了乙醇脱氢酶与NBD-TPEA的作用机理。实验结果发现不同结构和功能的蛋白对NBD-TPEA及NBD-TPEA-Zn的荧光产生不同程度的增敏影响,蛋白质中Trp残基的个数与其影响探针的荧光性质没有直接关系,其中ADH对探针的荧光产生明显的增敏效应,并计算了ADH与探针的结合常数及结合热力学类型,表明ADH与NBD-TPEA及NDB-TPEA-Zn的主要作用力为疏水、静电和氢键作用力；并用分子模拟的方法对其与ADH、CAH、GDH、Trypsin、HSA、γ-Globin、Lys的相互作用进行了研究,发现ADH中Tyr155与NBD环上的硝基形成氢键是ADH荧光增敏NBD-TPEA及NBD-TPEA-Zn可能的主要原因之一。这一结构研究有助于实现该类探针对ADH的荧光分子识别及针对不同蛋白质结构设计合成新型的能够选择性识别蛋白质的荧光探针。更多还原

【Abstract】 Proteins are the most important biomoleculars that play an important part in life hierarchy. The interactions between small drug molecular and proteins are important for molecular biology investigation. Human serum albumin (HSA) is the most abundant protein constituent of blood plasma and serves as a storage component protein. Human hemoglobin (Hb) is an oxygen-carrying protein.γ-Globin (γ-Gb), one of the most important immunoGlobin in organism, can also bind with drugs and antigen reversibly. In order to reduce the cellular resistance and systemic toxicity of cisplatin, some new platinum complexes have been designed and represented a new class of anticancer agents with distinctive cytotoxic activities and different mechanism of action. However, those new platinum complexes behavior towards proteins are totally unknown. Therefore, studies on the molecular details of such interactions could promote the understanding of platinum drugs in vivo and benefit the design of new platinum antitumor complexes.In this paper, on the basis of the previous research about the interactions of small molecular with proteins, the interactions between a monofunctional Pt (Ⅱ) complex(8-QA-Pt), a naphthalimide platinum complex (PEN-Pt), a dinuclear monofunctional Pt(Ⅱ) complex (Pt2-L) and a trinuclear monfunctional platinum(Ⅱ) complex (Pt3-L) and HSA, Hb and y-Gb were investigated by ultraviolet-visible(UV-Vis), Fourier transform infrared (FT-IR), circular dichroism (CD), and fluorescence spectroscopy, MALDI-TOF and molecular modeling methods. The results showed that the interaction mode of 8-QA-Pt, PEN-Pt, Pt2-L and Pt3-L with proteins were different from that of cisplatin. The associations between HSA (or Hb andγ-Gb) and 8-QA-Pt, PEN-Pt, and Pt3-L are most likely maintained through noncovalent interactions. However, the association between Pt2-L and HSA is most likely maintained through covalent interactions, the associations between Hb (andγ-Gb) are most likely noncovalent interactions. The binding constants, binding sites, and acting forces between 8-QA-Pt, PEN-Pt, Pt2-L and Pt3-L and proteins were discussed on the basis of fluorescence spectroscopic data. Chemical-induced protein denaturation and ligand competition experiments were performed to further identify the HSA primary binding site for 8-QA-Pt, PEN-Pt, Pt2-L and Pt3-L. The results show that 8-QA-Pt enters into the Subdomain IB, PEN-Pt, Pt2-L and Pt3-L partly enter subdomainⅡA. The binding regions and binding modes of the four platinum complexes on Hb andγ-Gb were obtained from the molecular modeling results. The effects of platinum complexes on the conformation of proteins were analyzed using UV-Vis, CD, FT-IR, synchronous fluorescence and three-dimensional fluorescence spectra. Noncovalent interactions with proteins are favorable for the availability of platinum drugs to tumor cells because they do not compromise the DNA binding ability of the drugs during delivery. In this case, proteins may function as an "unselfish" drug carrier and the protein-platinum complex system may serve as a drug reservoir for the therapeutic purpose. These results are beneficial to understanding the antitumor activity and toxicity of the monofunctional platinum (Ⅱ) complexes.Protein selective quantification is of fundamental importance in biochemical, biomedical research and disease treatment. As far as we know, few direct methods of detecting alcohol dehydrogenase (ADH) from Lactobacillus kefir quantity by fluorescence probes have been reported. In the paper, we used NBD-TPEA fluorescence probe to fluorescence detect ADH. The effects of human serum albumin, bovine serum albumin, lysozyme, trypsin,γ-Globin, carbonic anhydrase, glutamic dehydrogenase on the fluorescence of NBD-TPEA (of NBD-TPEA-Zn) were also studied. The results show that ADH enhances the fluorescence emission intensity of NBD-TPEA (of NBD-TPEA-Zn). The spectroscopic and thermodynamic data show that the interaction is a spontaneous process and the hydrogen bond, hydrophobic and ionic interactions are the primary contributors to the interaction between ADH and NBD-TPEA (or NBD-TPEA-Zn). The number of Trp in proteins is not the major reason of enhancing the fluorescence intensity of NBD-TPEA (or NBD-TPEA-Zn). The binding regions and binding modes of the NBD-TPEA (or NBD-TPEA-Zn) on proteins were also obtained from the molecular modeling method. The results show that the hydrogen bond between Tyr155 and NBD is mainly the reason of the fluorescence enhancing of NBD-TPEA (or NBD-TPEA-Zn). The remarkable fluorescence properties of NBD-TPEA help to extend the development of fluorescence probes for investigating enzymes in a biological context.更多还原