MCAT Practice Test 8

Titration of Histidine

¹Histidine is unusual among the amino acids because its side chain titrates near physiological pH. Histidine residues contribute much of the buffering capacity of proteins such as hemoglobin, and they often act as general acids or bases in enzyme active sites. Free histidine is also used as a buffer in some protein drug formulations, where it must hold the pH steady while the protein is stored for months.

²To characterize these acid–base properties, a student dissolved histidine in exactly two equivalents of hydrochloric acid to prepare 20.0 mL of 0.10 M histidine in its fully protonated form, which has a net charge of +2. The solution was titrated with 0.10 M NaOH at 25 °C, and the pH was recorded with a glass electrode calibrated with standard buffers at pH 4.00, 7.00, and 10.00. Each 0.25 equivalent of OH${}^{-}$ corresponds to 5.0 mL of titrant.

³The titration curve (Figure 1) shows three buffering regions. In order of increasing pH, they correspond to deprotonation of the α-carboxyl group, the side-chain group, and the α-amino group. Between buffering regions, the pH rises steeply at equivalence points, where nearly all of the histidine is in a single ionization state.

⁴The student planned to use the data for two purposes: to choose the composition of a histidine buffer for a protein that is most stable between pH 6.0 and 7.0, and to predict how histidine would migrate during electrophoresis at different pH values. Because a buffer resists pH change effectively only within about one pH unit of its pKa, the student expected only one of histidine's three ionizable groups to be useful for the formulation buffer.