Biospecific desorption from mixed-function columns.

enzymes. Necessary requirements include: (i) the enzyme must contain more than one ligand binding site; (ii) the bis-ligand should have strong affinity for the enzyme; (iii) the spacer connecting the two ligand entities should have sufficient length to cover the distance between two ligand binding sites situated on different molecules. AfRnity precipitation has been applied to several dehydrogenases such as lactate dehydrogenase, yeast alcohol dehydrogenase and glutamate dehydrogenase. Affinity precipitation of lactate dehydrogenase on a preparative scale from a crude extract is an easy procedure that rapidly yields pure enzyme (S. Flygare, T. Griffin, P.-0. Larsson & K. Mosbach, unpublished work). Affinity precipitation of enzymes resembles immunoprecipitation, i.e. the aggregation of antibodies and antigens. Immunological techniques such as immunodiffusion in agar gels (Ouchterlony test) can also be applied to enzymes-bis-ligands. It was shown that when lactate dehydrogenase and bis-NAD were allowed to diffuse towards each other in an agar gel containing pyruvate, a precipitation band was indeed formed. Modification of the agardiffusion technique allowed semiquantitative estimation of the amount of enzyme or of bis-NAD in samples of unknown composition.