In protein purification by chromatography, which property of the protein was exploited to separate it?

In protein purification by chromatography, which property of the protein was exploited to separate it?

• A. Position of hydrophobic R-groups
• B. Net charge of the protein
• C. Overall molecular weight
• D. Isoelectric point

Answer: (A)

Separation by this chromatography method hinges on how exposed hydrophobic surface areas are on the protein. Proteins have nonpolar, hydrophobic R-groups on their surfaces, and some have larger or more exposed hydrophobic patches than others. In a hydrophobic interaction chromatography setup, the stationary phase is nonpolar, so proteins with more hydrophobic surface interact more strongly and bind more tightly. By adjusting the solvent conditions—typically using high salt to promote binding and then lowering salt to weaken the interactions—the proteins elute in order of their hydrophobic character. More hydrophobic proteins stay attached longer and come off later, while less hydrophobic ones elute earlier. So, the property being exploited is the distribution and extent of hydrophobic R-groups on the protein surface. (Net charge and molecular weight are relevant in other chromatography methods, but hydrophobicity is what governs this one.)

Separation by this chromatography method hinges on how exposed hydrophobic surface areas are on the protein. Proteins have nonpolar, hydrophobic R-groups on their surfaces, and some have larger or more exposed hydrophobic patches than others. In a hydrophobic interaction chromatography setup, the stationary phase is nonpolar, so proteins with more hydrophobic surface interact more strongly and bind more tightly. By adjusting the solvent conditions—typically using high salt to promote binding and then lowering salt to weaken the interactions—the proteins elute in order of their hydrophobic character. More hydrophobic proteins stay attached longer and come off later, while less hydrophobic ones elute earlier. So, the property being exploited is the distribution and extent of hydrophobic R-groups on the protein surface. (Net charge and molecular weight are relevant in other chromatography methods, but hydrophobicity is what governs this one.)