Isolation and Purification

handle organisms with care gse bacteria without phage toxicity measured only by bioassay

The components of C2 toxin can be isolated from culture superna-tants of type C and type D Clostridium botulinum. It is important to remember that these organisms are capable of producing type C and type D botulinum neurotoxin. Although there are no documented cases of type C or type D poisoning in adults, recent work has shown that the isolated human neuromuscular ¡unction can be poisoned by type C toxin (Coffield and Simpson, unpublished). Therefore, extreme caution should be used when handling organisms that produce neurotoxin.

A simple alternative that greatly diminishes the hazard of isolating type C2 toxin is to use organisms from which bacteriophages have been eliminated. Both type C and type D toxin are encoded in phages that infect Clostridium botulinum (Hatheway, 1990). Therefore, eliminating the phages from the organisms removes the hazard associated with production of neurotoxin, but it does not alter growth and reproduction of the organisms, or their ability to make C2 toxin.

All investigators isolate the two components of C2 toxin by techniques originally described by Ohishi, Sakaguchi and their colleagues (Iwasaki et a/., 1980; Ohishi et al., 1980; Ohishi and Hama, 1992) (see Chapter 9). Although somewhat laborious, their method for isolating the toxin merely involves a series of chromatographic procedures. The heavy chain and light chain components do not appear in the same fractions, so different fractions must be combined to achieve full toxicity.

There are two techniques that can be used to confirm the identity of the isolated components: (a) cross-reactivity with chain-specific antibodies, and (b) bioassay. These techniques are equally easy to use, but the bioassay holds an enormous advantage. The immunologic technique confirms the presence of epitopes, but it reveals nothing about residual biological activity. Confirmation of toxicity can be achieved only by bioassay. Ideally, vulnerable cells should be exposed to toxin to evoke the characteristic rounding that results from loss of cytoskleleton, and substrate from poisoned cells should be isolated to confirm that cell rounding was indeed due to ADP-ribosylation.

If one is interested only in enzymatic modification of isolated substrate, then only the light chain component needs to be purified. This material can be used in the form that it is obtained at the final fractionation step. However, if one wishes to study toxin action on intact cells, both components must be isolated. Furthermore, the heavy chain component must be treated with trypsin before it will attain full biological activity. This can be achieved by mixing trypsin with heavy chain (1:10 on a protein basis) for 30min at 35°C at pH 7.5 (Ohishi, 1987). When run on polyacrylamide gels in the presence of sodium dodecyl sulfate, the native light chain migrates with an apparent molecular weight of approx. 50 000. The native heavy chain has an apparent molecular weight of approx. 100 000, whereas the activated heavy chain migrates with an apparent molecular weight of 88 000. In experiments involving intact cells, activated heavy chain is normally added at a two-fold molar excess to light chain.

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