Question

Suppose a column is filled with a cation-exchange resin at \(\mathrm{pH}\) 7.0. In what order would the given peptides elute from the column if each has the same number of residues? Peptide A: Ala \(30 \%\), Asp \(10 \%\), Lys \(10 \%\), Ser \(15 \%\), Pro \(25 \%\), Cys \(10 \%\) Peptide B: Ile \(25 \%\), Asp \(20 \%\), Arg \(5 \%\), Tyr \(15 \%\), His \(5 \%\), Thr \(30 \%\) Peptide C: Ala \(40 \%\), Glu 5\%, Arg 20\%, Ser 5\%, His 5\%, Trp \(25 \%\)

Short answer

Peptide B elutes first, followed by A, then C.

Step-by-step solution

Determine Ionization of Amino Acids at pH 7

At pH 7, acidic amino acids like Aspartic acid (Asp) and Glutamic acid (Glu) will generally carry a negative charge, and basic amino acids like Lysine (Lys), Arginine (Arg), and Histidine (His) can carry a positive charge. However, at pH 7, His is not fully ionized.

Calculate Net Charge for Each Peptide

For Peptide A, basic residue is Lys (10%) and acidic residue is Asp (10%). This translates to a net neutral charge as both cancel out. For Peptide B, basic residue is Arg (5%) and acidic residue is Asp (20%). Thus, net charge is negative. For Peptide C, basic residues are Arg (20%) and acidic residue is Glu (5%). This gives a net positive charge.

Determine Elution Order from the Cation-Exchange Column

In a cation-exchange column, positively charged peptides bind more strongly. Order is determined from most negative (elute first) to most positive (elute last). Peptide B (negative charge) elutes first, then Peptide A (neutral charge), and Peptide C (positive charge) elutes last.

Key concepts

Cation-Exchange Chromatography

Cation-exchange chromatography is a widely-used technique for separating molecules. It is particularly useful in protein purification. The principle behind cation-exchange chromatography involves the interaction between a resin in the column and charged molecules, like peptides and proteins.

In this method, the resin contains negatively charged groups. These groups interact and bind with positively charged particles, also known as cations. This is why positively charged peptides bind more strongly to the cation-exchange column.

During the protein purification process, when a sample is passed through the column, peptides with different net charges will move at different rates. Knowing the net charge at a particular pH helps predict the order in which different peptides will elute. The most negatively charged peptides will elute first, as they have the weakest interaction with the negatively charged resin. Conversely, the most positively charged peptides will elute last because they have the strongest binding to the resin. This ordered separation enables the purification of peptides based on their charge characteristics.

Peptide Ionization

Peptide ionization is a crucial factor in understanding how peptides interact within a chromatographic system. Ionization refers to the process in which a molecule, like a peptide, gains or loses an electron, resulting in a net positive or negative charge.

Amino acids that compose peptides can be classified based on their side chain properties as acidic, basic, or neutral. Acidic amino acids like aspartic acid and glutamic acid tend to lose protons in a neutral pH environment, thus giving them a negative charge. Basic amino acids such as lysine, arginine, and histidine can gain protons, contributing to a positive charge. However, each amino acid has a characteristic pH, known as its isoelectric point (pI), where the amino acid is neutral.

When dealing with peptide ionization at a specific pH, like pH 7 in the exercise, it is important to consider which residues are ionized and contribute to the net charge of the peptide. This ionization state will directly influence how the peptide behaves in cation-exchange chromatography by dictating its binding affinity to the resin and thus its elution order.

Amino Acid Charge

The charge of an amino acid is determined by its side chain. This charge is pivotal in predicting the behavior of peptides in chromatic conditions. At any given pH, the ionizable side chains of amino acids will exhibit distinct charged states that affect the overall charge of a peptide.

- **Acidic amino acids (e.g., Aspartic acid and Glutamic acid):** These generally have a negative charge at a neutral pH of 7 because they tend to lose protons.
- **Basic amino acids (e.g., Lysine, Arginine, Histidine):** These typically carry a positive charge because they can gain a proton.
- **Neutral amino acids (e.g., Alanine, Glycine):** These do not contribute to the net charge as their side chains do not ionize at physiological pH.

Understanding the charge properties of these amino acids allows you to determine the net charge of a peptide under different conditions. In the example provided, calculating the net charge involves assessing the percentages of each charge-contributing amino acid present in the peptide and the pH of the solution. This information is crucial for guiding peptides through cation-exchange chromatography effectively and predicting their elution order.