Ligand-Protein Coprecipitative Isolation by Matrix Stacking and Entanglement

• Published in: Separation science and technology 2000-01, Vol.35 (11), p.1795-1811
• Main Authors: LOVRIEN, REX, WU, CHARLES, MATULIS, DAUMANTAS
• Format: Article
• Language: English
• ISSN: 0149-6395; 1520-5754
• DOI: 10.1081/SS-100102494

Ligands are being developed for the upstream isolation-purification of sought-for proteins from dilute crudes by ligand-protein coprecipitation. The ligands are alkane-substituted azoaromatic anions (dyes) with sulfonate heads. Overall coprecipitation is comprised of two main reactions. Ligands first bind electrostatically and stoichiometrically to protein molecule cationic side chains in solution, approximately to a point where the protein net charge Z H + is ion-pair titrated with organic anion ligand heads. Organic tail groups cover a sizable portion of the protein molecular surface, triggering 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. Protein molecule structural determinants for coprecipitation of a sought-for protein are protein cationic charge density and location (governed by pH, amino acid composition, and Scatchard-Black reactions). Ligand structural determinants for forcing coprecipitation using 10 −5 to 10 −4 M ligands depend on the ion pairing capacity of the ligands (which determines the stoichiometry) and the details and size of the organic moiety of the ligands. Binding ligands to the target protein in solution contributes the initial part of the overall coprecipitation. However ligand-ligand interactions, in conjunction with ligand placement on proteins to build the host lattice, contribute a large part of the overall coprecipitation. They are sharply dependent on the foregoing factors and on the topology of each lattice to determine the selectivity of matrix ligand coprecipitation. An example is presented of direct coprecipitation of two lectins out of their crudes. Very strongly acting ligands that sweep most proteins and polypeptides out of solution are available. However, use of the maximal coprecipitating power is not necessarily the best strategy. Rather, there needs be struck a balance between coprecipitating power, selectivity, and reversibility for later release of the sought-for protein.