Ion-exchange chromatography in the presence of the non-ionic dissociating agent chloral hydrate. Application to a membrane protein, bovine heart cytochrome c oxidase.

In previous work we have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) is a potent non-ionic protein dissociating agent. We have employed it in systems of polyacrylamide-gel electrophoresis and have demonstrated the presence of 15 components in a preparation of bovine heart cytochrome c oxidase [Griffin & Landon (1981) Biochem. J. 197, 333-344]. Here we describe the use of solutions containing aq. 100% (w/v) chloral hydrate in the ion-exchange column chromatographic separation on CM-cellulose of the alpha- and beta-chains of human haemoglobin, which we have employed as a model protein of known structure. We also describe the use of similar procedures in order to fractionate the polypeptide components of bovine heart cytochrome c oxidase. An effective separation has been obtained and we suggest that chloral hydrate-containing solutions could have general application in the ion-exchange-chromatographic analysis of membrane proteins, a procedure that has had restricted use owing to the inadequacy of non-ionic dissociating agents available previously.