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| 水母毒素蛋白凝聚现象的初步研究 |
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于华华1,2, 刘希光1,2, 邢荣娥1,2, 刘松1,2, 李鹏程1
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1.中国科学院海洋研究所 青岛266071;2.中国科学院研究生院北京100039
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| 摘要: |
| 通过测定水母毒素的凝聚速度,来初步研究水母毒素蛋白的凝聚现象,这为水母毒素蛋白的提取利用及研究毒素蛋白的结构、稳定性、生物活性等提供重要参考依据。实验结果表明,在0.01mol/L,pH6的磷酸缓冲溶液中,0.003mol/L的Vc、EDTA、苯甲酸均能降低水母毒素蛋白的凝聚速度;蔗糖对水母毒素的抗凝聚现象不明显。 |
| 关键词: 水母 毒素蛋白 凝聚现象 |
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| 基金项目:中国科学院重要方向性项目,KZCX3-SW-215号;青岛市科技资助项目,02-1-KJ-SHN-24号 |
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| PRELIMINARY STUDY ON AGGLOMERATION OF JELLYFISH VENOM PROTEIN |
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YU Hua-Hua1,2, LIU Xi-Guang1,2, XING Rong-E1,2, LIU Song1,2, LI Peng-Cheng1
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1.Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071;2.Graduate School, Chinese Academy of Sciences, Beijing, 100039
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| Abstract: |
| Jellyfish belongs to coelenterate and is also called Cnidaria whose cells have been differentiated into simple muscle, netro-tissue and unique nematocysts. Jellyfish widely distributes from the South China Sea, the Yellow Sea to the Bohai Sea. About 400 species of jellyfish have been recorded in China. Jellyfish is a rich source of marine biology in distribution and species. However, it has been used only as food after being simply processed. Jellyfish stings human body and releases venom, causing burning pain, erythematous eruptionsand and itching. Syndromes include fever, fatigue, muscle aches, tight breath, dropsy, blood press depression and even death. So, it is essential to study jellyfish venom to turn harmful venom into beneficial medicine for human heath purpose, as scientist did for snake venom and balloonfish venom.
Jellyfish venom is a special type of protein; it has many bioactivities such as enzymatic activities, hemolysis, hepatocyte toxicity, myotoxicity, cardiac toxicity and netro-toxicity. However, further study on jellyfish venom has been complicated by many factors such as thermal instability, aggregation of heterogeneous proteins and peptides, the presence of protease during purification, and different species. Up to present, the study on jellyfish venom has been mainly focused on purification, structure characterization, bioactivity determination, and the cure of the sting to human being. The integrated primary structure of jellyfish venom protein (JVP) has not been reported so far, and regarding bioactivity assay is currently in animal experiment stage. Study on agglomeration of JVP has not been reported yet. During the venom agglomeration, disulfide bonds form between molecules. Because disulfide bond is one of the act ion forces that maintain the tertiary structure of JVP; the JVP would degenerate with protein agglomeration resulting in some bioactivities loss. In this paper, the effect of different solutions on the agglomeration of JVP was studied by measuring agglomeration velocity; and the optimal conditions for JVP stabilization was explored.
Jellyfish Rhopilema esculentum kishinouye used in the experiment were collected from a pier in Shazikou area, Qingdao City, Shandong Province, China, in September 2001. Tentacles were manually excised in vivo, and frozen immediately at -20°C. The frozen tentacles were homogenated and then sonicated in different cold (4°C) solutions for four times at 15s each at interval 30s. The resultant fluids were centrifuged at 13000 rpm for 20min at 4°C; and the supernatant was used as JVP solutions. The JVP solutions were deposited at 4°C for protein agglomerating. Then centrifuged it at 16000 rpm for 20 min at 4°C and determined the concentration of protein in the supernatant. Agglomeration velocity V(mg/ml*h)=(C0-Ct)/t, C0: the JVP concentration at time zero, Ct: the JVP concentration at test time, t: deposition time.
The results show that agglomeration velocity of JVP was reduced in phosphate buffer solution (0.01 mol/L, pH 6) containing vitamin C (0.003mol/L). Perhaps H+ in Vc made-SH reductive in JVP and prohibited disulfide bond from forming, while the role of the Vc needs future in-depth study. EDTA in three concentrations (0.001 mol/L, 0.003 mol/L, 0.005 mol/L) can reduce the agglomeration velocity. It was due to two reasons. One was that EDTA chelated with metal ions such as Mg2+, Ca2+ and Mn2+, resulting in decrease in stabilization of the tertiary structure. The other is that the activity of JVP-bearing metalloprotease was inhibited due to EDTA chelating with metal ion that is essential for metalloprotease activity. No obvious difference in the effect among the treatments in three concentrations of EDTA. It is probably that the concentrations of metal ions in JVP were so low that 0.001mol/L EDTA could chelate them completely. Benzoic acid could also reduce the agglomeration because it sterilized the JVP solution. However, sucrose is a co-solvent and can change the thermodynamic feature of a solution. A balance can be established when hydration of protein surface completes and combines the co-solvent entirely. The balance would stabilize the natural protein but for reducing protein agglomeration, the effect was weak. |
| Key words: Agglomeration, Jellyf ish,Venom protein |
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