Preparation of complement fragments C3b and C3a from purified rat complement component C3 by activated cobra venom factor.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Usami
Makoto
M
Division of Pharmacology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan. usami@nihs.go.jp
Ohno
Yasuo
Y
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods; D003176:Complement C3 / Q000737:chemistry*; D015926:Complement C3a / Q000032:analysis / Q000302:isolation & purification*; D003179:Complement C3b / Q000032:analysis / Q000302:isolation & purification*; D004546:Elapid Venoms / Q000737:chemistry* / Q000302:isolation & purification; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013678:Technology, Pharmaceutical / Q000379:methods
D003176:Complement C3; D004546:Elapid Venoms; C016310:cobra venom factor; D015926:Complement C3a; D003179:Complement C3b