11] Diphtheria toxin: Purification and properties

Abstract

Publisher Summary This chapter describes the purification and properties of diphtheria toxin (DT). The chapter also discusses the purification of DT from toxin preparations obtained from commercial sources, and its subsequent fractionation into the various forms. Monomeric DT is a complex enzyme that performs three functions during the course of intoxicating sensitive mammalian cells. It binds to receptors on the cell surface; undergoes an internalization and translocation process whereby at least the enzymatically active portion of the toxin penetrates to the cytosolic compartment; and catalyzes transfer of the adenosine diphosphate (ADP)-ribosyl moiety of nicotinamide adenine dinucleotide (NAD) into covalent linkage with elongation factor 2 (EF-2), blocking protein synthesis and causing cell death. Appropriate choice of ionic strength and elution conditions resolves the culture supernatant into monomeric, dimeric, and other oligomeric forms of DT. A variable percentage of the toxin in these supernatants is already nicked, and the remainder is generally converted to the nicked form by treatment with low concentrations of trypsin.