Quantitative analysis of protein: immobilized dye interaction.

The interaction of rabbit muscle lactate dehydrogenase with reactive blue 2 immobilized on Sepharose CL-6B was quantitatively evaluated by analytical procedures using both frontal and zonal chromatography and also static equilibrium methodology. All three analytical procedures give a common result, namely (i) that each enzyme molecule is retained by a single immobilized dye molecule; (ii) that the immobilized dye binds to the protein at an NADH binding site with an affinity identical to that of the free dye; and (iii) that less than 2% of the immobilized dye is accessible to the enzyme. The very small fractional accessibility is not due to steric exclusion but likely is a result of adsorption of the immobilized dye to the matrix surface.