光谱法结合化学计量学研究石墨烯量子点与胰蛋白酶的相互作用

Interaction Between Graphene Quantum Dots and Trypsin With Spectroscopic and Chemometrics Approaches

石墨烯量子点(GQDs)以其优异的性能在生物医学领域引起了广泛的关注,但其潜在的毒性研究较少。将荧光和紫外光谱法结合化学计量学研究GQDs对胰蛋白酶结构和功能的影响。从荧光猝灭实验可知,GQDs可猝灭胰蛋白酶的固有荧光并抑制胰蛋白酶的生物活性。当加入不同浓度的GQDs,胰蛋白酶在350 nm处的荧光发射峰的强度随之降低且蓝移(350~344 nm),表明GQDs可改变胰蛋白酶所处的微环境,使其疏水性增加;与此同时,GQDs浓度越高,胰蛋白酶荧光变化越明显,说明GQDs对胰蛋白酶可能有潜在毒性。通过圆二色谱实验可知胰蛋白酶的α-螺旋结构由19.12%下降至16.23%,说明GQDs加入诱导胰蛋白酶的二级结构发生改变,使胰蛋白酶骨架松弛;三维荧光光谱实验进一步说明GQDs的存在不仅改变了胰蛋白酶所处的微环境并使胰蛋白酶的构象发生变化。蛋白质氨基酸残基的微环境由蛋白质分子的构象所决定,当蛋白质的生色团所处的微环境发生变化时,其紫外-可见吸收光谱也随之发生变化。由于生命作用体系都比较复杂,测量所得到的波谱数据中大部分信息是隐含和重叠的,因此需要利用和发展有效的生物信号采集、转导、数据处理和解析方法,把能对生命现象做出解释的有用信息尽可能多地从测量数据中挖掘出来。为获取足够而有效的生命化学信息,该研究用连续滴定技术采集多维光谱数据,运用多元曲线分辨-交替最小二乘法(MCR-ALS)解析光谱数据矩阵,从重叠严重的光谱中同时得到定性(各组分光谱及作用过程中复合物的真实存在)和定量(各组分的浓度变化趋势)信息,从而认识GQDs与胰蛋白酶在作用中达到平衡时各组分的状态和整个动态变化过程。MCR-ALS的解析结果为进一步了解GQDs与胰蛋白酶相互作用的动力学过程提供了依据,说明GQDs可以与胰蛋白酶相互作用,并形成GQDs 15-胰蛋白酶复合物。该研究为GQDs可能存在的毒性风险研究提供了信息。

With super properties,such as photoluminescence properties,edge effect,low cytotoxicity and great biocompatibility,graphene quantum dots(GQDs)have attracted great attention in biological and biomedical applications.The potential toxicity investigations of GQDs still need to involve.Few studies have been illuminated that GQDs could alter the function and structure of trypsin.The molecular interaction between trypsin and GQDs was systematically researched through the combination of multi-spectroscopic and chemometrics approaches.The fluorescence quenching experiment showed that GQDs quench the intrinsic fluorescence of trypsin and inhibit the biological activity of trypsin.When different concentrations of GQDs were added,the fluorescence emission peak intensity of trypsin at 350 nm continuously decreased and had a blue shifted(350 to 344 nm),indicating that GQDs could change the microenvironment of trypsin and increase its hydrophobic.Meanwhile,the higher the concentration of GQDs,the more obvious the change of trypsin fluorescence,indicating that GQDs interacts and changes the secondary structure of the macromolecule.The microenvironment of protein amino acid residues is determined by the conformation of protein molecules.The spiral structure of proteinase decreased from 19.12%to 16.23%in the circular dichroism experiment indicated that the addition of GQDs induced the alteration of the secondary structure of trypsin and relaxed the trypsin framework.The three-dimensional fluorescence further indicated that the conformation of trypsin changed with the addition of GQDs.When the microenvironment of the chromophores of serum albumin changes,its UV-visible absorption spectrum also changes.Due to the complex life system,most of the information in the test of spectrum data is implicit and overlapping.We need to use and develop the effective biological signal collection,transduction,data processing and analysis method to get the useful information that can explain life as much as possible from the measured data.To obtain sufficient and effective chemical information of life,this study adopts the continuous titration technique collecting multidimensional spectrum data.An expanded UV-Vis spectral data matrix was analyzed by the multivariate curve resolution-alternating least squares(MCR-ALS)chemometrics approach.To further understand the state and the whole dynamic change process of each component when GQDs and trypsin reached equilibrium in action,the qualitative(spectrum of each component)and quantitative(the changing trend of concentration)information were obtained from the heavily overlapped spectra.The analytical results of MCR-ALS provide a basis for further understanding of the kinetic process of the interaction between GQDs and trypsin,indicating that GQDs can interact with trypsin and form GQDs 15-trypsin complex.The results offered insights into the binding mechanism of GQDs with trypsin and significant information for possible toxicity risk of GQDs to human health.