The physical properties of two isoforms of a protein, both of which bind the same ligand, are shown in the table: Which protocol would yield the best separation of the two isoforms?

Question

The physical properties of two isoforms of a protein, both of which bind the same ligand, are shown in the table:  Which protocol would yield the best separation of the two isoforms?

Quizplus · Accepted Answer

11ecba2d_0ec6_8b5e_a3bf_6b7e2ba2b741_MD0008_00 Separation techniques take advantage of molecular properties to cause some molecules to move faster than others through a stationary phase. As the molecules exit the stationary phase (elute), they can be collected at different time points. Column chromatography is a set of techniques for separating proteins by charge (ion exchange), molecular weight (size exclusion), or binding partner (affinity). The charge of a protein depends on the pH at which the protein has a net charge of zero, which is known as the isoelectric point (pI). When the ambient pH is lower than the pI, the protein gains protons and becomes positively charged. When pH is greater than the pI, the protein loses protons and becomes negatively charged. Both isoforms shown in the table have pI values below physiological pH (7.4), so at this pH, both are negatively charged and will bind to an anion-exchange column. When NaCl is added, the chloride anions compete with the negatively charged proteins for binding. Because Isoform 1 has a lower pI than Isoform 2, it is more negatively charged and binds the column more tightly. Therefore, Isoform 2 will elute at a lower salt concentration than Isoform 1. Gradually increasing the salt concentration in the buffer allows each isoform to be collected separately. (Choice A) Cation-exchange columns have negatively charged beads that bind positively charged molecules and repel negatively charged ones. At pH 10, both isoforms are negatively charged and would be repelled by the beads, exiting the column together. (Choice B) Both isoforms are approximately the same size (~68 kDa), so they will elute from the size-exclusion column together. (Choice D) Both isoforms bind the same ligand with nearly identical affinity (~3 × 10⁻⁶ M), so both would bind to the affinity column. This technique would separate the two isoforms from other proteins but not from each other. Educational objective: Proteins are often purified on chromatography columns. Ion-exchange columns separate molecules by charge (cation-exchange columns bind cations; anion-exchange columns bind anions), size-exclusion columns separate molecules by size, and affinity columns isolate molecules that bind a particular ligand.

The Physical Properties of Two Isoforms of a Protein, Both