Question

a) Draw the complete covalent structure of the tripeptide Met-Tyr-Arg, with trans peptide bonds, as it would exist at pH 7.

b) Write the 1-letter code for each of the three amino acids below the structure.

c) What is the overall charge of this peptide at pH 7?

d) What is the overall charge at pH 12?

e) This tri-peptide is reacted with 6M HCl and heat, which breaks-up the individual peptide bonds. The mixture of the 3 amino acids is then run through a cation-exchange column. Which of the amino acids will come out first, which second and which third?

Answer 1

#e. Note the following points-

I. Cation exchange chromatography (matric is negatively charged) separates cations (positively charged proteins, or other molecules) based on their relative charges. At a pH below (more acidic) its pI, the protein is positively charged. Greater is the difference between pH of medium and pI of the protein, larger fraction of the protein is positively charged.

II. In cation exchange chromatography, the protein or amino acids with greater positive charge is retained on the negatively charged matrix of stationary more strongly. So, a protein with maximum positive charged in eluted last.

III. Isoelectric point (pI) is the pH at which an amino acid exists in electrically neutral species.

At pH = pI : No net gain or loss of proton from the specified amino acid.

At pH < pI : (pH below pI): The carboxylate ( COO-) and amine (NH2) group (if any) of the specified amino acid accepts proton to counteract increase in pH. Protein is positively charged.

At pH > pI : (pH above pI): The carboxyl acid (COOH) and amine (NH3+) group donates proton to counteract decrease in pH. Protein is negatively charged.

IV. pI of met = 5.74 , pI of Tyr = 5.66       , pI of Arg =10.76

Since the peptide digested in 6M HCl, the resultant solution is extremely acidic, say near 1.0. Lower is the pI (acidic pH) of an amino acid, lower is the net charge (closer to zero) on it. So, the residue of lowest pI would be eluted first, and the amino acid with highest pI would be eluted last.

So, order of elution (first to last) is: Tyr > Met > Arg

#d.

# Calculation of Charge: Note the following points-

I. Only the C-ter, N-ter and ionizable side chains (acidic and basic residues) are used to calculate net charge on the peptide.

II. The charge is calculated using Henderson-Hasselbalch equation. A sample calculation is shown at the bottom.