Usami Makoto, Ohno Yasuo
Division of Pharmacology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan.
J Pharmacol Toxicol Methods. 2005 Sep-Oct;52(2):260-3. doi: 10.1016/j.vascn.2004.12.001.
Complement component C3 (C3) can be a target of pharmacological or toxicological agents. In the analysis of this, it is important to examine the involvement of fragments C3b and C3a since C3 function normally requires cleavage into these fragments. The present study describes a simple and efficient method for the preparation of rat complement C3b and C3a by using purified C3 and cobra venom factor (CVF) as a cleaving enzyme.
CVF was purified from lyophilized cobra venom (Naja naja kausia) by two-step chromatography and was activated by incubation with human factors B and D. C3 was cleaved by incubation with activated CVF (CVF,Bb), and C3b and C3a were isolated by anion- and cation-exchange chromatography, respectively.
About 200 microg of CVF was purified from 100 mg of cobra venom. All the CVF was activated by incubation with factors B and D. The C3b and C3a obtained were pure as analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and no digestive by-products such as C3f were found.
The advantage of the present method is that it is possible to prepare relatively large amounts of C3b by simple procedures without digestive by-products. C3a can be prepared from the flow through fraction of the C3b purification. C3b and C3a prepared by the present method would be useful for pharmacological or toxicological experiments involving receptor binding since their binding sites remain intact.
补体成分C3（C3）可能是药理或毒理制剂的作用靶点。在对此进行分析时，检查C3b和C3a片段的参与情况很重要，因为C3的正常功能需要裂解成这些片段。本研究描述了一种简单有效的方法，通过使用纯化的C3和眼镜蛇毒因子（CVF）作为裂解酶来制备大鼠补体C3b和C3a。
通过两步色谱法从冻干的眼镜蛇毒（眼镜蛇）中纯化CVF，并通过与人因子B和D孵育进行激活。通过与活化的CVF（CVF，Bb）孵育裂解C3，分别通过阴离子和阳离子交换色谱法分离C3b和C3a。
从100mg眼镜蛇毒中纯化出约200μg CVF。所有CVF通过与因子B和D孵育而被激活。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，获得的C3b和C3a是纯的，未发现诸如C3f等消化副产物。
本方法的优点是可以通过简单的程序制备相对大量的C3b，且无消化副产物。C3a可以从C3b纯化的流穿组分中制备。通过本方法制备的C3b和C3a对于涉及受体结合的药理或毒理实验将是有用的，因为它们的结合位点保持完整。
