Dihydrofolate reductases from chicken liver and Lactobacillus casei bind Cibacron blue F3GA in different modes and at different sites.
The binding of Cibacron blue F3GA to dihydrofolate reductase (EC 1.5.1.3) from chicken liver an amethopterin-resistant Lactobacillus casei has been studied by difference spectroscopy. The blue dye binds to the enzyme from each species in a specific fashion with a 1:1 stoichiometry. However, the mode of interaction of the dye with the enzyme and the site of interaction on the enzyme are very different for the avian and bacterial enzymes. The dye seems to bind in an almost totally "electrostatic mode" at the dihydrofolate binding site to the chicken liver enzyme and is displaced from the enzyme only by dihydrofolate, folate, or methotrexate and not at  all by NADPH. In contrast, the binding of the dye to the bacterial enzyme is characterized by a totally "apolar interaction" at a site partially overlapping both the NADPH site and the methotrexate/dihydrofolate site. NADPH can displace the dye only partially and methotrexate is more efficient than NADPH in displacing the dye. Both NADPH and methotrexate are needed for a total displacement of the dye from the bacterial enzyme. We propose that the blue dye is capable of binding specifically to any protein possessing a cluster of aromatic and other apolar groups and/or geometrically spaced positively charged groups for proper interaction with the aromatic rings and/or sulfonate groups of the dye molecule. The so-called specificity of the blue dye for the nucleotide binding proteins is thus a special case of the above mentioned requirements and not diagnostic of the "dinucleotide fold."
D000818:Animals; D000873:Anthracenes; D001665:Binding Sites; D002645:Chickens; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D011485:Protein Binding; D013045:Species Specificity; D013053:Spectrophotometry; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014227:Triazines
D000873:Anthracenes; D014227:Triazines; C012842:Cibacron Blue F 3GA; D013762:Tetrahydrofolate Dehydrogenase