MCAT practice · Chemical and Physical Foundations · 5A Water, acids and bases · 5D Biomolecule structure
Titrating carnosine, a muscle dipeptide
An original practice passage written for this site, not an AAMC item. Exam pace is about 8 minutes for a passage like this.
Carnosine (β-alanyl-L-histidine) is a dipeptide present at millimolar concentrations in vertebrate skeletal muscle. During intense exercise, protons released by glycolysis and ATP hydrolysis can lower intracellular pH from about 7.1 to about 6.5, and this acidification is thought to contribute to fatigue. A research group asked how well carnosine resists that fall in pH and how it compares with related compounds.
In Experiment 1, the researchers dissolved carnosine to 10.0 mM in 20.0 mL of water and added HCl until all three of its ionizable groups were protonated: the C-terminal α-carboxyl group of the histidine residue, the imidazole side chain, and the amino group of the β-alanine residue. They then titrated the solution with 0.100 M NaOH at 25 °C, recording the pH after each addition. The curve, corrected for base consumed by the solvent alone, is shown in Figure 1 as equivalents of OH⁻ added per mole of carnosine.
In Experiment 2, the researchers prepared 20.0 mM solutions, each adjusted to pH 7.10, of four compounds: carnosine; anserine, a carnosine analog carrying a methyl group on the imidazole ring that is abundant in bird muscle; free L-histidine; and β-alanine, the N-terminal residue of carnosine. Each solution was titrated with HCl until its pH reached 6.50, a range chosen to match the fall seen in working muscle. The H⁺ absorbed by each compound was calculated after subtracting the acid needed to move a water blank across the same range. Each measurement was made in triplicate, and the results are given in Table 1.
The researchers noted that anserine and carnosine behaved similarly in Experiment 2, whereas the other two compounds did not, and they planned further work on the methylated analog.
| Compound | H⁺ absorbed (mM) | SD (mM) |
|---|---|---|
| Carnosine | 6.6 | 0.2 |
| Anserine | 6.3 | 0.2 |
| L-Histidine | 3.3 | 0.1 |
| β-Alanine | 0.1 | 0.1 |
Data are illustrative, constructed for practice.
1. Based on Figure 1, the isoelectric point of carnosine is closest to which pH?
- A. Wrong: 4.7 is the average of the first two pKa values (2.6 and 6.8). The form between them carries a net charge of +1, not zero, so these are the wrong pKa values to average.
- B. Wrong: 6.8 is the imidazole pKa, the midpoint of the second plateau. At that pH, half the molecules are +1 and half are 0, so the average charge is +0.5.
- C. Right: the net-zero form (−COO⁻, neutral imidazole, −NH₃⁺) exists between the second and third plateaus. Average the pKa values on either side of it: (6.8 + 9.4)/2 = 8.1.
- D. Wrong: 9.4 is the amino-group pKa. At that pH, half the molecules are 0 and half are −1, so the average charge is −0.5.
2. A sample of carnosine is run on an electrophoresis gel buffered at pH 7.4. Based on Figure 1, carnosine will most likely:
- A. Right: pH 7.4 is below the pI of about 8.1, so the net charge is positive. The carboxyl group is −1 and the amino group is about +1. The imidazole is 0.6 unit above its pKa of 6.8, so only about 20% of it is protonated. The net charge is about +0.2, a slow drift toward the negative electrode, the cathode.
- B. Wrong: a charge near +1 requires a fully protonated imidazole. At pH 7.4, above the imidazole pKa of 6.8, most imidazole groups have lost their proton.
- C. Wrong: this treats carnosine like glycine, which is a neutral zwitterion at physiological pH. With its third ionizable group, carnosine's pI is about 8.1, not 7.4.
- D. Wrong: this reverses the rule. A molecule is net negative only at a pH above its pI, and 7.4 is below carnosine's pI of about 8.1.
3. The researchers assigned the plateau near pH 6.8 in Figure 1 to the imidazole side chain. Which additional experiment would most directly test this assignment?
- A. Wrong: a back-titration tests whether protonation is reversible. The same three groups would give the same three plateaus, so the assignment stays untested.
- B. Wrong: mobility measures net charge, which comes from all three groups together. It cannot tell you which group gives up its proton near pH 6.8.
- C. Wrong: doubling the concentration doubles the base each plateau consumes, but the pKa values stay put. It cannot show which group causes which plateau.
- D. Right: β-alanyl-alanine has the same peptide backbone, terminal carboxyl and terminal amino group as carnosine, with alanine's methyl side chain in place of histidine's imidazole side chain. If its curve has no plateau near pH 6.8, that plateau belongs to the imidazole. The comparison changes one variable.
4. According to Figure 1, after 2.5 equivalents of OH⁻ have been added, which two forms of carnosine are present in roughly equal amounts?
- A. Wrong: this pair is equal at 0.5 equivalent, the midpoint of the first plateau near pH 2.6, where the carboxyl group is half titrated.
- B. Right: at 2.5 equivalents the pH is 9.4, the midpoint of the third plateau. The carboxyl and imidazole groups have already given up their protons, and the amino group is half deprotonated.
- C. Wrong: this pair is equal at 1.5 equivalents, near pH 6.8, where the imidazole is half titrated.
- D. Wrong: this has the amino group losing its proton before the imidazole. Groups give up protons in order of pKa, and the imidazole (about 6.8) goes before the amino group (about 9.4).
5. Table 1 shows that free L-histidine absorbed about half as much H⁺ as carnosine. If the imidazole pKa of free histidine is about 6.0, which statement best explains this difference?
- A. Right: by Henderson-Hasselbalch, about 7% of histidine's imidazole is protonated at pH 7.1 and about 24% at pH 6.5. Only about 17% of it takes up a proton (about 3.3 mM of 20 mM). Carnosine's pKa of 6.8 sits mid-window, so about 33% of its imidazole takes up a proton (about 6.6 mM).
- B. Wrong: this is the sign error. The whole range is above pH 6.0, and above its pKa a group is mostly deprotonated, not protonated.
- C. Wrong: free histidine and carnosine each have three ionizable groups (α-carboxyl, imidazole, amino). They have the same number of sites.
- D. Wrong: an α-carboxyl group has a pKa near 2. At pH 6.5 to 7.1 it is fully deprotonated and takes up essentially no protons.
Missed questions come back in your review queue after 1, 3, 7 and 21 days. More passages →