Zonal chromatographic analysis of the interaction of alcohol dehydrogenase with blue-sepharose.

The interaction between horse liver alcohol dehydrogenase and Reactive blue 2 immobilized on Sepharose CL-6B was measured by zonal chromatography. Each protein molecule was retained by a single immobilized dye using a Blue-Sepharose column containing a total of 1.38 mM dye. However, the protein was predominantly retained by two immobilized dye molecules using a darker Blue-Sepharose column containing a total of 11.6 mM dye. The dissociation constant measured for the alcohol dehydrogenase--immobilized dye complex on each column is identical to the inhibition constant for the alcohol dehydrogenase--free Reactive blue 2 complex: 4.5 +/- 0.8 microM.