光疗药物—竹红菌甲素与血红素类蛋白质的相互作用研究
摘要
本课题所研究的竹红菌甲素(HA)是一种具有应用前景的天然光疗药物。它通过静脉注射的方式进入人体，在此过程中，HA会与血液的各种成分发生作用，从而使其结构和性质发生改变。因此，通过研究HA与血液中血红素类蛋白质之间的相互作用，探讨HA在人体血液中的传输机理，对于进一步推动竹红菌素类光疗药物在光动力疗法领域中的应用发展具有重要意义。
本论文用紫外—可见吸收光谱、荧光光谱、顺磁共振等方法研究了HA与溶菌酶、肌红蛋白和血红蛋白的相互作用情况。结果表明HA与蛋白质的表面氨基酸残基发生作用，作用方式是表面结合，对蛋白质内部结构如卟啉环没有产生直接影响。它们之间的作用力类型主要是静电力和疏水力，该作用使得蛋白质构象发生明显改变。通过实验结果可以判断HA与蛋白质之间形成了复合物，所有蛋白质荧光猝灭过程均为伴随着电子传递的静态猝灭，同时求算出它们之间相互作用的结合常数和结合位点数，以及形成复合物过程中的热力学和动力学参数。实验证明蛋白质中激发态氨基酸残基与HA之间没有发生直接的能量传递，原因可能是由于HA的吸收光谱与蛋白质的荧光光谱重叠太小。
另外利用拉曼光谱研究了HA光敏损伤溶菌酶的微观结构变化。其本质是HA产生的活性氧(~1O_2，O_2~(-·)和·OH等)破坏了溶菌酶的有序结构，使溶菌酶主链结构的α-螺旋、β-折叠、β-回折明显减少，无规卷曲增加，使其侧链结构的吲哚环、二硫键和碳硫键明显减少，并且使色氨酸残基所处微环境以及二硫链中碳原子构象发生变化，说明溶菌酶的主链构象发生了变化。
Hypocrellin A (HA), which was studied in this paper, is a potential phototherapeutic drug. In its photodynamic therapeutic procedure, it is injected intravenously into the patients. During this period, HA will react with many components of blood, and change their structures and properties. So it is important to study the interaction between HA and hemachrome protein and discuss the transport mechanism of HA in body for the development of hypocrellin apllication in the field of photodynamic therapy.
The interaction between HA and some kinds of protein (such as lysozyme, myoglobin and hemoglobin) was studied by spectrophotometer, spectrofluorimeter and ESR spectrometer. It was found that the binding occured on the surface of protein through surface amino acid residues, and changed the conformation of protein, and had no direct effect on its interior structure. Their interaction forces are hydrophobic and electrostatic forces. Based on the experimental data, it was concluded that there are compounds forming between HA and protein, and all fluorescence quenchings are static with electron transfer. We can gain the binding constants and binding sites by dwelling with data, as well as the thermodynamic values and dynamic values during the formative process of the compound. The experiments show that there is no nonradiative energy transfer occurring between HA and the excited state of protein, it can be due to the small overlap between the fluorescence emission spectra of protein and the absorption spectra of HA.
The results of Laser Raman spectroscopy indicated that active oxygen species (~10_2, O_2~- and OH etc.) generated by HA can photodamage the structure of lysozyme. The orderly conformation (such as a-helix, p-sheet and P-turn) decreased obviously while the random coil increased. The indole ring, disulfide bond and carbon-sulfur bond in the side chain of lysozyme also decreased obviously. The change of the micro-environment of tryptophane residue and the carbon conformation of the disulfide chain in lysozyme shows that the conformation of the main chain changed.
本论文用紫外—可见吸收光谱、荧光光谱、顺磁共振等方法研究了HA与溶菌酶、肌红蛋白和血红蛋白的相互作用情况。结果表明HA与蛋白质的表面氨基酸残基发生作用，作用方式是表面结合，对蛋白质内部结构如卟啉环没有产生直接影响。它们之间的作用力类型主要是静电力和疏水力，该作用使得蛋白质构象发生明显改变。通过实验结果可以判断HA与蛋白质之间形成了复合物，所有蛋白质荧光猝灭过程均为伴随着电子传递的静态猝灭，同时求算出它们之间相互作用的结合常数和结合位点数，以及形成复合物过程中的热力学和动力学参数。实验证明蛋白质中激发态氨基酸残基与HA之间没有发生直接的能量传递，原因可能是由于HA的吸收光谱与蛋白质的荧光光谱重叠太小。
另外利用拉曼光谱研究了HA光敏损伤溶菌酶的微观结构变化。其本质是HA产生的活性氧(~1O_2，O_2~(-·)和·OH等)破坏了溶菌酶的有序结构，使溶菌酶主链结构的α-螺旋、β-折叠、β-回折明显减少，无规卷曲增加，使其侧链结构的吲哚环、二硫键和碳硫键明显减少，并且使色氨酸残基所处微环境以及二硫链中碳原子构象发生变化，说明溶菌酶的主链构象发生了变化。
Hypocrellin A (HA), which was studied in this paper, is a potential phototherapeutic drug. In its photodynamic therapeutic procedure, it is injected intravenously into the patients. During this period, HA will react with many components of blood, and change their structures and properties. So it is important to study the interaction between HA and hemachrome protein and discuss the transport mechanism of HA in body for the development of hypocrellin apllication in the field of photodynamic therapy.
The interaction between HA and some kinds of protein (such as lysozyme, myoglobin and hemoglobin) was studied by spectrophotometer, spectrofluorimeter and ESR spectrometer. It was found that the binding occured on the surface of protein through surface amino acid residues, and changed the conformation of protein, and had no direct effect on its interior structure. Their interaction forces are hydrophobic and electrostatic forces. Based on the experimental data, it was concluded that there are compounds forming between HA and protein, and all fluorescence quenchings are static with electron transfer. We can gain the binding constants and binding sites by dwelling with data, as well as the thermodynamic values and dynamic values during the formative process of the compound. The experiments show that there is no nonradiative energy transfer occurring between HA and the excited state of protein, it can be due to the small overlap between the fluorescence emission spectra of protein and the absorption spectra of HA.
The results of Laser Raman spectroscopy indicated that active oxygen species (~10_2, O_2~- and OH etc.) generated by HA can photodamage the structure of lysozyme. The orderly conformation (such as a-helix, p-sheet and P-turn) decreased obviously while the random coil increased. The indole ring, disulfide bond and carbon-sulfur bond in the side chain of lysozyme also decreased obviously. The change of the micro-environment of tryptophane residue and the carbon conformation of the disulfide chain in lysozyme shows that the conformation of the main chain changed.
引文
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