Ligand-protein coprecipitative isolation by matrix stacking and entanglement

Ligands are being developed for the upstream isolation-purification of soug ht-for proteins from dilute crudes by ligand-protein coprecipitation. The l igands are alkane-substituted azoaromatic anions (dyes) with sulfonate head s. Overall coprecipitation is comprised of two main reactions. Ligands firs t bind electrostatically and stoichiometrically to protein molecule cationi c side chains in solution, approximately to a point where the protein net c harge Z(H+) is ion-pair titrated with organic anion ligand heads. Organic t ail groups cover a sizable portion of the protein molecular surface, trigge ring the second category of reactions; matrix formation and coprecipitation . Organic tails stack and hydrophobically associate, pulling the complexes together in a host lattice or matrix, enclosing protein molecule guests. Pr otein molecule structural determinants for coprecipitation of a sought-for protein are protein cationic charge density and location (governed by pH, a mino acid composition, and Scatchard-Black reactions). Ligand structural de terminants for forcing coprecipitation using 10(-5) to 10(-4) M ligands dep end on the ion pairing capacity of the ligands (which determines the stoich iometry) and the details and size of the organic moiety of the ligands. Bin ding ligands to the target protein in solution contributes the initial part of the overall coprecipitation. However ligand-ligand interactions, in con junction with ligand placement on proteins to build the host lattice, contr ibute a large part of the overall coprecipitation. They are sharply depende nt on the foregoing factors and on the topology of each lattice to determin e the selectivity of matrix ligand coprecipitation. An example is presented of direct coprecipitation of two lectins out of their crudes. Very strongl y acting ligands that sweep most proteins and polypeptides out of solution are available. However, use of the maximal coprecipitating power is not nec essarily the best strategy. Rather, there needs be struck a balance between coprecipitating power, selectivity, and reversibility for later release of the sought-for protein.