Complications encountered using cibacron blue F3G-A as a ligand for affinity precipitation of lactate dehydrogenase

The use of the affinity interaction between Cibacron Blue F3G-A (CB) and NADH-dependent enzymes to selectively precipitate these enzymes has been examined. An attempt was made to form crosslinked precipitates of lactate dehydrogenase (LDH) using bis- and poly-CB conjugates. When precipitation was not observed, an examination of the interaction between the enzyme and the conjugated CB was made. Quasielastic light scattering indicated only a slight radius increase, the greatest being from 50 to 130 A, when a CB-dextran conjugate was added to a solution of LDH, and no increase when bis-CB made with a 1, 6-diaminohexane spacer was added to a similar solution. The results of enzyme inhibition studies showed that conjugated CB bound at the NAD+ site of LDH. Spectral measurements of the conjugated CB below 5 μM were similar to those reported for a stacking interaction that occurs in solutions with CB concentrations above 5 μM We conclude that the conjugated CB is binding to the LDH, but that a competing dye stacking interaction prevents extensive cross-linking of the LDH, and thus inhibits precipitation.