Abstract
This chapter summarizes recent successful applications of specific enzyme–substrate interaction to the purification of enzymes. It also describes two modified procedures that have been developed for the large-scale purification of rabbit liver fructose-1,6-diphosphatase (FDPase). One of these methods utilizes elution of the enzyme from carboxymethyl (CM)–cellulose by a concave salt gradient containing dilute substrate. This approach may be of general applicability in enzyme purification wherein the combined effects of increasing ionic strength and the affinity of a substrate, activator, or inhibitor for the enzyme bring about selective enzyme elution. Homogeneous FDPase is obtained in large quantities by a single adsorption and elution from CM–cellulose. The purification of rabbit liver fructose-1,6-diphosphatase is investigated in the chapter. Some of the fractions eluted from CM–cellulose initially have slightly higher specific activities (24–30 units/mg protein) than those reported for homogeneous enzyme (20–22.5 units/mg protein). However, after several freezings and thawings, the specific activities generally dropped to 22–23.
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