Principles of multienzyme purifications by affinity chromatography

Abstract

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.