Related Experiment Videos
Dimeric form of diphtheria toxin: purification and characterization
Biochemistry
|May 6, 1986
Summary
Dimeric diphtheria toxin (DT) is largely inactive because it cannot bind cell receptors. However, it can regain toxicity by dissociating into active monomeric forms, especially in vivo.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Diphtheria toxin (DT) preparations often contain monomeric and dimeric forms.
- Understanding the distinct properties of these forms is crucial for toxin research.
Purpose of the Study:
- To compare the biochemical and toxicological properties of purified monomeric and dimeric DT.
- To elucidate the mechanism behind the observed differences in toxicity.
Main Methods:
- Fractionation of DT into monomeric and dimeric forms (>98% purity).
- In vitro assays: dissociation kinetics, ADP-ribosyltransferase, NAD-glycohydrolase, and ligand-binding assays.
- In vivo assays: rabbit skin tests and guinea pig lethality tests.
Main Results:
- Dimeric DT slowly dissociates to active monomers in solution and rapidly with DMSO.
- Dimeric DT showed minimal toxicity in cell culture and rabbit skin tests, attributed to monomer contamination or dissociation.
- Higher doses of dimeric DT were required to cause lethality in guinea pigs compared to monomeric DT, with delayed onset.
Conclusions:
- Dimeric DT is primarily non-toxic due to impaired receptor binding, likely caused by structural changes in the B moiety.
- The observed in vivo toxicity is linked to slow dissociation into active monomeric units.
- Hydrophobic interactions between B moieties likely stabilize the dimeric form, occluding the receptor-binding site.