MCAT practice · Biological and Biochemical Foundations · 1A Proteins and enzymes · 3B Organ systems
Why fetal hemoglobin holds oxygen more tightly
An original practice passage written for this site, not an AAMC item. Exam pace is about 8 minutes for a passage like this.
A fetus must take up oxygen from maternal blood across the placenta, where the two circulations exchange gases without mixing. Fetal red cells contain mainly hemoglobin F (HbF, α₂γ₂), whereas adult red cells contain hemoglobin A (HbA, α₂β₂). In whole blood, fetal cells bind O₂ with higher affinity than adult cells. The two proteins differ at several positions; one of them is residue 143, a histidine that lines the central cavity between the two β chains of HbA but a serine in the γ chains of HbF. A research group asked whether the affinity difference lies in the proteins' intrinsic O₂ affinity or in how each protein responds to the red-cell metabolite 2,3-bisphosphoglycerate (2,3-BPG).
HbA and HbF were purified from adult and umbilical-cord blood and passed over an ion-exchange column to remove bound organic phosphates, giving "stripped" protein. Each protein (60 µM tetramer) was studied in 50 mM HEPES buffer with 0.1 M KCl at 37 °C. Samples were equilibrated in a tonometer with gas mixtures of known pO₂, and fractional O₂ saturation was calculated from the visible absorbance spectrum. Each protein was measured stripped and with 2 mM 2,3-BPG added, a large molar excess over hemoglobin. HbA with 2,3-BPG was measured at pH 7.4 and again at pH 7.2; all other conditions were at pH 7.4. P50 and the Hill coefficient (n_H) were obtained by fitting each curve to the Hill equation, using four independent preparations per condition.
Stripped HbA and stripped HbF had nearly the same P50 (Table 1). Adding 2,3-BPG shifted both curves to the right, but the shift for HbA was almost twice the shift for HbF (Figure 1). Lowering the pH of the HbA sample from 7.4 to 7.2 shifted its curve further to the right. For interpreting the curves, the authors took the pO₂ of blood leaving the lungs as about 100 torr and the pO₂ in active tissue as about 20 torr.
- Stripped HbA, pH 7.4
- HbF + 2,3-BPG, pH 7.4
- HbA + 2,3-BPG, pH 7.4
- HbA + 2,3-BPG, pH 7.2
| Sample | pH | P50 (torr) | n_H |
|---|---|---|---|
| Stripped HbA | 7.4 | 12.1 ± 0.4 | 2.6 ± 0.1 |
| Stripped HbF | 7.4 | 11.8 ± 0.5 | 2.6 ± 0.1 |
| HbA + 2,3-BPG | 7.4 | 26.5 ± 0.8 | 2.8 ± 0.1 |
| HbF + 2,3-BPG | 7.4 | 19.6 ± 0.6 | 2.7 ± 0.1 |
| HbA + 2,3-BPG | 7.2 | 33.0 ± 1.0 | 2.8 ± 0.1 |
Data are illustrative, constructed for practice.
1. Which mechanism best explains the shift of the HbA curve when the pH is lowered from 7.4 to 7.2?
- A. Wrong: dissociated αβ dimers bind O₂ with high affinity and little cooperativity, which would shift the curve left and lower n_H; n_H stays at 2.8.
- B. Wrong: oxidation to Fe³⁺ makes methemoglobin, which cannot bind O₂ at all; a 0.2 pH unit drop does not do this, and the curve still reaches near-full saturation.
- C. Right: at lower pH, histidines such as β146 are protonated and form salt bridges (with Asp β94) that hold the low-affinity T state, shifting the curve to the right.
- D. Wrong: this is the common misreading of the Bohr effect. Protons bind the protein away from the heme; they lower affinity allosterically, not by competing at the iron.
2. Based on Figure 1, about what percentage of HbA's O₂-binding sites give up their O₂ as blood moves from the lungs to active tissue at pH 7.2 with 2,3-BPG present?
- A. Wrong: about 20% is the saturation remaining at 20 torr on the pH 7.2 curve, not the amount released.
- B. Wrong: about 43% is the saturation at 30 torr on the pH 7.2 curve, a misread of the tissue pO₂.
- C. Wrong: about 66% is the unloading for HbA with 2,3-BPG at pH 7.4 (97.6% − 31.3%), read from the wrong curve.
- D. Right: on the pH 7.2 curve saturation is about 96% at 100 torr and about 20% at 20 torr, so about 76% of the sites unload.
3. The authors conclude that HbF binds O₂ more tightly in red cells mainly because it binds 2,3-BPG weakly. Which result, had it been observed, would most weaken this conclusion?
- A. Right: if the stripped proteins already differed and 2,3-BPG shifted both equally, the affinity difference would lie in the proteins' intrinsic O₂ affinity, not in 2,3-BPG binding.
- B. Wrong: this is essentially what was observed, and it supports the conclusion rather than weakening it.
- C. Wrong: equal cooperativity is consistent with either explanation, so it cannot weaken the conclusion.
- D. Wrong: a similar Bohr effect in both proteins says nothing about whether they differ in 2,3-BPG binding.
4. Suppose a fetus carried a γ-chain variant in which serine 143 is replaced by histidine, with no other change. Compared with normal HbF, the variant in fetal red cells would most likely show:
- A. Right: histidine restores a positive charge that helps bind the anionic 2,3-BPG, so the variant should respond more like HbA, with a right-shifted curve and less O₂ loaded at the placenta.
- B. Wrong: the similar stripped P50 values show the two proteins have similar intrinsic O₂ affinity; the mutation acts on 2,3-BPG binding, which red cells contain, so P50 in cells would change.
- C. Wrong: this reverses the effect. Adding a positive charge to the central cavity should strengthen 2,3-BPG binding and lower affinity, raising P50.
- D. Wrong: 2,3-BPG stabilizes the T state but does not abolish cooperativity; n_H stayed near 2.7–2.8 with 2,3-BPG in Table 1.
5. Red cells stored for transfusion lose most of their 2,3-BPG. If such cells behave like stripped HbA in this study, then right after transfusion, compared with normal adult cells, they would deliver about how much O₂ between the lungs and active tissue?
- A. Wrong: this assumes higher affinity means better delivery. Stripped HbA loads fully in the lungs but stays about 79% saturated at 20 torr, so it unloads less.
- B. Wrong: both are nearly saturated at 100 torr, but delivery depends on how much is released at 20 torr, where the curves differ greatly.
- C. Wrong: this is the ratio of the P50 values (12.1/26.5 ≈ 0.46), which is not the same as the ratio of O₂ released.
- D. Right: stripped HbA releases about 99.6% − 78.7% ≈ 21% of its sites, versus about 97.6% − 31.3% ≈ 66% for HbA with 2,3-BPG, a ratio of about 0.3.
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