Coprecipitation of proteins with matrix ligands: Scaleable protein isolation

Abstract

Matrix ligands are agents for isolating proteins out of dilute crudes by coprecipitating proteins. The ligands have a strong anion sulfonate head which initiates binding to proteins having a positive net charge, ZH+ approximately 5-20. Initial binding tightens protein conformation and starts to squeeze water from conformationally motile proteins. The tails are stackable hydrophobic organic groups, azoaromatic dyes which draw protein-ligand complexes together. Proteins coprecipitate as guests, in the ligand host matrix. In addition to stacking, ligand tails displace water because of their bulk, and lower the average dielectric constant near charged groups, which reinforces the electrostatic component of binding. Matrix ligands protect proteins, scavenge them from dilute crudes (0.01-0.1 per cent protein), and densify coprecipitates. Detergent ions in low concentrations, 10(-4)-10(-5) M also sometimes serve as coprecipitating agents, entangling their tails but probably not stacking. Divalent metal ions, Zn++, sometimes are useful auxiliary agents. Preparative scaleability from crudes is demonstrated starting from 100-200 g of raw peanuts and raw pineapple to coprecipitate a lectin and bromelain enzyme respectively in 1-2 h with 80-90 per cent activity yields. Ligands are released from coprecipitates by shifting the pH and trapping the ligands with exchange resins. Protein conformation tightening in solution is seen by viscosity measurements.

1-Anilino-8-Naphthalene Sulfonate Anion-Protein Binding Depends Primarily on Ion Pair Formation

“…However, the stoichiometry over the broad range of ANS Ϫ binding, from sparse binding up to saturation, is determined by the sulfonate group of ANS Ϫ in conjunction with the number of cationic (histidine, lysine, arginine side chains) groups donated by the protein molecule. This behavior is similar, nearly congruent, to that of other kinds of organic sulfonate and sulfate ligands able to vigorously bind to protein molecules, such as detergent sulfates and azoaromatic dye sulfonates of many kinds (Matulis et al, 1996;Conroy and Lovrien, 1992). The dominantly electrostatic determinant, the origin, of ANS Ϫ stoichiometry is indicated by the sharp pH dependency of the ANS Ϫ binding process.…”

1-Anilino-8-Naphthalene Sulfonate Anion-Protein Binding Depends Primarily on Ion Pair Formation

“…However, the stoichiometry over the broad range of ANS Ϫ binding, from sparse binding up to saturation, is determined by the sulfonate group of ANS Ϫ in conjunction with the number of cationic (histidine, lysine, arginine side chains) groups donated by the protein molecule. This behavior is similar, nearly congruent, to that of other kinds of organic sulfonate and sulfate ligands able to vigorously bind to protein molecules, such as detergent sulfates and azoaromatic dye sulfonates of many kinds (Matulis et al, 1996;Conroy and Lovrien, 1992). The dominantly electrostatic determinant, the origin, of ANS Ϫ stoichiometry is indicated by the sharp pH dependency of the ANS Ϫ binding process.…”

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

“…The primary binding mechanism of the fluorophore, 1-anilino-8-naphthalene sulfonate (ANS), is through ion pair formation with lysine, arginine and histidine residues [37]. Similar behavior has been observed with other organic sulfonate and sulfate ligands, including detergent sulfates such as SDS [38,39]. Collectively, these observations suggest that basic amino acids play a fundamental role in binding anionic detergent and are therefore crucial to SDS-mediated interactions of hydrophobic fluorophores such as SYPRO Tangerine dye.…”

Ligand–Protein Coprecipitative Isolation by Matrix Stacking and Entanglement

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1-Anilino-8-Naphthalene Sulfonate Anion-Protein Binding Depends Primarily on Ion Pair Formation

“…However, the stoichiometry over the broad range of ANS Ϫ binding, from sparse binding up to saturation, is determined by the sulfonate group of ANS Ϫ in conjunction with the number of cationic (histidine, lysine, arginine side chains) groups donated by the protein molecule. This behavior is similar, nearly congruent, to that of other kinds of organic sulfonate and sulfate ligands able to vigorously bind to protein molecules, such as detergent sulfates and azoaromatic dye sulfonates of many kinds (Matulis et al, 1996;Conroy and Lovrien, 1992). The dominantly electrostatic determinant, the origin, of ANS Ϫ stoichiometry is indicated by the sharp pH dependency of the ANS Ϫ binding process.…”

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

“…The primary binding mechanism of the fluorophore, 1-anilino-8-naphthalene sulfonate (ANS), is through ion pair formation with lysine, arginine and histidine residues [37]. Similar behavior has been observed with other organic sulfonate and sulfate ligands, including detergent sulfates such as SDS [38,39]. Collectively, these observations suggest that basic amino acids play a fundamental role in binding anionic detergent and are therefore crucial to SDS-mediated interactions of hydrophobic fluorophores such as SYPRO Tangerine dye.…”

Ligand–Protein Coprecipitative Isolation by Matrix Stacking and Entanglement

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1-Anilino-8-Naphthalene Sulfonate Anion-Protein Binding Depends Primarily on Ion Pair Formation

“…However, the stoichiometry over the broad range of ANS Ϫ binding, from sparse binding up to saturation, is determined by the sulfonate group of ANS Ϫ in conjunction with the number of cationic (histidine, lysine, arginine side chains) groups donated by the protein molecule. This behavior is similar, nearly congruent, to that of other kinds of organic sulfonate and sulfate ligands able to vigorously bind to protein molecules, such as detergent sulfates and azoaromatic dye sulfonates of many kinds (Matulis et al, 1996;Conroy and Lovrien, 1992). The dominantly electrostatic determinant, the origin, of ANS Ϫ stoichiometry is indicated by the sharp pH dependency of the ANS Ϫ binding process.…”

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

“…The primary binding mechanism of the fluorophore, 1-anilino-8-naphthalene sulfonate (ANS), is through ion pair formation with lysine, arginine and histidine residues [37]. Similar behavior has been observed with other organic sulfonate and sulfate ligands, including detergent sulfates such as SDS [38,39]. Collectively, these observations suggest that basic amino acids play a fundamental role in binding anionic detergent and are therefore crucial to SDS-mediated interactions of hydrophobic fluorophores such as SYPRO Tangerine dye.…”

Ligand–Protein Coprecipitative Isolation by Matrix Stacking and Entanglement

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