A procedure is described to utilize blue dextran-Sepharose as an affinity chromatographic column specific for the super-secondary structure called the dinucleotide fold, which forms the binding sites for substrates and effectors on a wide range of proteins. The procedure can be used to identify proteins, either purified or in crude cellular extracts, that possess the dinucleotide fold and to significantly improve the purification procedures for those proteins that possess the fold.A sulfonated polyaromatic blue dye covalently attached to dextran, called blue dextran,O-DEXTRAN is commonly used to measure the void volume of exclusion chromatographic columns. It has been observed that several proteins that normally penetrate the internal volume of exclusion gel beads are totally excluded from the internal volume when chromatographed along with blue dextran in the presence of low, but not high, ionic strength solvents. A variety of proteins exhibit this behavior, such as pyruvate kinase (1, 2), glutathione reductase (3), and blood coagulation factors II, VII, IX, and X (4). It was concluded that blue dextran forms a complex with each of these proteins that is dissociable by salt. Subsequent to these observations, blue dextran-Sepharose affinity columns have been made and used in the final purification stages of enzymes such as phosphofructokinase (5) and lactate dehydrogenase (6).These enzymes were displaced from the affinity columns by addition of low concentrations of their nucleotide substrates to the elution solvents.We propose that the blue dextran complexes with this wide range of proteins because it is specific for a super-secondary structure called the dinucleotide fold. This structure involves about 120 amino acids that are arranged in a ,-sheet core composed of five or six parallel strands connected by ahelical intrastrand loops located above and below the ,8-sheet (7,8). The dinucleotide fold is known to form the NADbinding site in lactate (9), malate (10), and glyceraldehydephosphate (11) dehydrogenases, to form the ATP-binding site in phosphoglycerate kinase (12), and to be present in the structures of alcohol dehydrogenase (13), adenylate kinase (14,15), and phosphoglycerate mutase (16). In addition, the FMN-binding site of flavodoxin (17,18) and the aromatic specificity site in the proteolytic enzyme subtilisin (19,20) have been proposed (7,8) to contain a remnant of the dinucleotide fold termed the mononucleotide fold. Accordingly, we examined the interaction of blue dextran-Sepharose affinity columns with each of these proteins as well as with several proteins known not to possess the dinucleotide fold to determine the specificity of the affinity columns.
MATERIALS AND METHODS
