A simple procedure is described for determination of the amount of covalently bound Cibacron blue F3GA dye present in preparations of blue Sephadex and blue dextran-Sepharose. This method involves hydrolysis of the Sephadex and Sepharose dye derivatives in 6 M hydrochloric acid followed by spectrophotometric determination of the dye released.
The preparation of blue dextran-Sepharose and blue Sephadex and their uses in general ligand affinity chromatography have recently been described (l-3). They have been employed in the preparation of a wide variety of dehydrogenases and kinases [see particularly Ref. ( 2)]. Both of these affinity media contain a covalently bound polyaromatic blue dye known as Cibacron blue F3GA. The molecular dimensions of this blue chromophore closely resemble those of the cofactor NAD. It has been found that proteins possessing the supersecondary structure known as the dinucleotide fold will bind to Sepharose and Sephadex dye derivatives. These compounds may be conveniently prepared in large quantities in the laboratory. In order to compare results of experiments employing different batch preparations of these media it is important to know the degree of substitution of Cibacron blue F3GA on the gel matrix.
This paper describes a method developed from procedures suggested by Easterday and Easterday (3) by which this may be achieved. The method involves liberation of the bound ligand from the matrix by acid hydrolysis followed by spectrophotometric determination of the amount of dye released.
Methods
Sephadex G-150 (40-120 pm) and blue dextran were purchased from Pharmacia Fine Chemicals. Cibacron F3GA (Polysciences, Inc.) was used to prepare blue Sephadex G-150 by the method of Easterday and Easterday (3). Blue dextran-Sepharose 4B, prepared by the method of Ryan and Vestling (l), was generously provided by Dr. M. E. Knuth. All other chemicals used were reagent grade or better.