Removal of thioctic acid from enzymes.

The r81e of thioctic acid in t’he oxidative decarboxylation of or-keto acids is now well established (1, 2). This was accomplished largely by studies with microbial cells grown essentially free of the cofactor. In some instances (2) it has been possible t,o separate the coenzyme from microbial enzymes involved in t,hioctic acid metabolism by extensive purification procedures. Although tjhe direct demonstration of the function of thioctic acid in vertebrate enzyme systems is lacking, t,he occurrence of large amounts of the cofactor in purified pyruvic (3) and oc-ketoglutaric (4) oxidases from pigeon breast muscle and pig heart indicates (5) that the rBle of the cofactor in vertebrate systems parallels that in microbes. The factor is bound so tightly to the vertebrate enzymes that such procedures as dialysis, repeated washings, or ion exchange techniques do not dislodge it. Experiments with the oc-keto acid oxidases from the ciliated protozoan, Tetrahymena pyriformis S, indicate that thioctic acid can be readily removed from the enzyme by adsorption with alumina (6). It has since been recognized (7) that cofactor removal by this procedure requires the participation of an enzyme fraction which seems to catalyze the liberation of thioctic acid from protein-bound coenzyme in a reversible manner; the free thioctic acid which is liberated is then adsorbed on the alumina. Ammonium sulfate precipitation of pigeon liver acetone powder extracts separates a fraction which liberates the cofactor from enzymes. Since this fraction has not as yet been purified enough to ascertain the nature and specificity of the enzyme, the ammonium sulfate fraction has been termed (8) a “thioctic acid-splitting” fraction which contains “thioctic acid-splitting” activity.