Covalent Modification Reactions Involving Cytosolic Aldehyde Dehydrogenase and a Variety of Resorufin Derivatives

Abstract

The bromoacetates of resorufin and ofp-nitrophenol act simultaneously as very rapidly hydrolyzed substrates and as potent irreversible inactivators of cytosolic aldehyde dehydrogenase. Following inactivation, the resorufin orp-nitrophenoxide moiety is released fairly quickly by hydrolysis from the modified enzyme. Resorufin dimethylcarbamate and resorufin methanesulphonate inactivate aldehyde dehydrogenase in an “oxidative addition” reaction that is consistent with the involvement of the enzyme's catalytically essential cysteine residue with the quinonimine ring of the resorufin structure. The resulting covalently modified enzyme is yellow-orange and the redox properties of the colored label can be conveniently observed. Resorufin ethyl ether also slowly inactivates aldehyde dehydrogenase, but the resulting labeled enzyme is a surprising pink-violet color at pH values of about 5 to 10. The expected yellow-orange derivative is obtained at pH less than 4 or upon denaturation of the protein. The possible chemical identity of the species responsible for the pink-violet color is discussed. The resorufin anion binds noncovalently to aldehyde dehydrogenase apparently at the cofactor binding site, as it may be displaced by NAD+or NADH.