C.2.3

=Explain how proteins can be analyzed by chromatography and electrophoresis.= Primary structure (chain of amino acids) of proteins can be determined either paper chromatography or by electrophoresis. First, protein must be **hydrolysed** with **concentrated hydrochloric acid**, HCl to **break the peptide bond** between the amino acids thus separating them. The 3D structure of the complete protein can be confirmed by X-ray crystallography.

Paper chromatography
1. Heat the protein to a __high temperature__ with __concentrated HCl__ to hydrolyze the protein / break it down into individual amino acids / break the peptide bonds. 2. A small spot of the unknown amino acid mixture is placed near the bottom of a piece of chromatographic paper. 3. Separate spots of known amino acids can be placed alongside. 4. The paper is placed in water (solvent) which then rises up the paper due to capillary action. 5. As the solvent meets the sample spots, the different amino acids move up the paper at different rates due to their differing solubilities in the solvent. 6. When the solvent has nearly reached the top, the paper is removed from the tank, dried, and then sprayed with a dye (ninhydrin) to make the amino acids visible. 7. The Rf value (retention factor) of the unknown spots is then compared with the Rf value of the known amino acids.

The retention factor is the ratio of the distance the amino acid travels compared to the distance the solvent travels. The Rf value is a good indicator of the identity of an unknown amino acid. If the Rf value of the unknown and the known amino acid are close to one another then the two are likely to be the same.

Image: Chemistry for the IB Diploma, Neuss, OUP, 2001

Amino Acid Equilibrium
An equilibrium exists when an amino acid is in an aqueous solution. Consider the equilibrium for glycine given below.



The structure of glycine changes at different pH levels. At its **isoelectric point:** the pH value, which is unique for each amino acid, at which an amino acid carries no net electrical charge and exists as a zwitterion (a molecule that carries both positive and negative charges) - the charge on the amino acid is 0. This is because its charges are balanced because the amino acid has a positive NH3+ group and a negative charge COO- group. Each amino acid has a unique isoelectronic point, and because of this an individual amino can be identified. Table 20 of the Chemistry data booklet has a table of amino acids and their pH at their isoelectric point.

At a **acidic pH below the isoelectric point pH** the amine group acts as a bronsted-lowry base (think acid base reaction with the buffer as the acid and the amino acid as the base). The amino acid gains a proton (H+) from the acid. The amino acid becomes **positively charged**. (Structure C).

H2N-CH2-COOH + H+ ---› +H3N-CH2-COOH


 * At high basic pH** **above the isoelectric pH** the carboxylic acid group acts as a bronsted-lowry acid and loses a proton to the base.The amino acid becomes **negatively charged**. (Structure A). Structure B is called a zwitterion. The amino group has gained a proton and the carboxyl group has lost a proton giving an amino acid with both a positive and negative charge.

H2N-CH2-COOH ---› H2N-CH2-COO**-** + H+

Electrophoresis
1. Heat the protein to a high temperature with concentrated HCl to hydrolyze the protein / break it down into individual amino acids / break the peptide bonds. 2. The amino acid sample is placed in the centre of the gel that is placed in a buffer solution of known pH and a potential difference / voltage applied between the positive anode and negative cathode (as in electrolysis). The gel containing the amino acids is placed in a buffer solution. The buffer keeps the pH constant so as to prevent an change in the charge on the amino acids. 3. Depending on the pH of the buffer the different amino acids will move at different rates. If the **pH of the buffer is less than the isoelectric point** **pH** of the amino acid, the buffer solution acts as the acid and the amino acid as the base and will accept a proton. The **positive ion** will predominate and it will **move towards the negative cathode**. If the **pH of the buffer is greater than the isoelectric point pH** of the amino acid, the buffer will act as a base and the amino acid as an acid and donate a proton. A **negative ion** will predominate and it will **move towards the positive anode**. If the pH of the buffer is equal to the isoelectric point, the amino acid will not move. 4. When the amino acids are separated the gel is developed with ninhydrin to make the amino acids visible. 5. The distances the amino acids have traveled can be compared with standing values or compared with their isoelectric points. Image: Chemistry for the IB Diploma, Neuss, OUP, 2001

Consider the following example: The amino acids arginine, cysteine and glycine undergo electrophoresis at a pH of 6.0. Identify the direction that the animo acids will travel on the gel. 1. Find the pH of each amino acid at its isoelectric point: arginine pH 10.8, cysteine pH 5.1 and glycine pH 6.0. 2. Compare the pH at the isoelectric point with the pH of the solution. For arginine pH at IE 10.8 › 6.0, for cysteine pH at IE 5.1 ‹ 6.0 and for glycine pH at IE 6.0 = 6.0**.** Therefore arginine 6.0 is less than the pH at the isoelectric point and so positive ion will predominate which will move towards the negative cathode. For cysteine 6.0 is greater than the pH at the isoelectric point and so the negative ion will predominate and move towards the positive anode. for glycine the pH of the buffer is equal to the isoelectric point, so it will not move.