📚 Haemoglobin | 血红蛋白
Haemoglobin is a globular, conjugated protein found in red blood cells (erythrocytes). It serves as the primary respiratory pigment in vertebrates, responsible for transporting molecular oxygen (O₂) from the lungs to respiring tissues and facilitating the return transport of carbon dioxide (CO₂). Its unique quaternary structure and cooperative binding behaviour are central to its vital physiological role.
血红蛋白是存在于红细胞中的一种球状结合蛋白。它是脊椎动物体内主要的呼吸色素，负责将分子氧(O₂)从肺部运送到正在进行呼吸的组织，同时促进二氧化碳(CO₂)的返回运输。其独特的四级结构和协同结合行为对于这一关键生理功能至关重要。
1. Introduction: The Respiratory Pigment | 引言：呼吸色素
Haemoglobin accounts for approximately 95% of the dry mass of an erythrocyte and imparts the characteristic red colour to blood. Each haemoglobin molecule is a tetramer — an assembly of four polypeptide chains — each of which non‑covalently binds a haem prosthetic group containing a ferrous iron (Fe²⁺) ion. It is the iron that reversibly binds one molecule of O₂, enabling the protein to function as a molecular oxygen carrier.
血红蛋白约占红细胞干重的95%，并赋予血液特有的红色。每个血红蛋白分子是一个四聚体——由四条多肽链组装而成——每条链都以非共价方式结合一个含二价铁离子(Fe²⁺)的血红素辅基。正是这个铁原子可逆地结合一分子O₂，使该蛋白成为分子氧的载体。
Because haemoglobin must load O₂ in the lungs, where the partial pressure of oxygen (pO₂) is high, and then release it in the tissues, where pO₂ is low, its oxygen‑binding properties must be precisely regulated. This regulation is achieved through a combination of allosteric control, cooperative subunit interactions and environmental factors such as pH and CO₂ concentration.
由于血红蛋白必须在氧气分压(pO₂)高的肺部装载O₂，然后在pO₂低的组织中将其释放，其氧结合特性必须受到精确调控。这种调控通过变构控制、亚基协同作用以及pH和CO₂浓度等环境因素的共同作用来实现。
2. Quaternary Structure: Globin Chains and Haem | 四级结构：珠蛋白链与血红素
Adult haemoglobin (HbA) is a tetramer composed of two α‑globin and two β‑globin polypeptide chains, giving it the subunit composition α₂β₂. Each globin chain is folded into a specific tertiary structure that contains a hydrophobic pocket precisely shaped to accommodate the haem group. The four subunits are held together by non‑covalent interactions — predominantly hydrogen bonds, hydrophobic contacts and a small number of ionic interactions — forming a compact, roughly spherical quaternary assembly.
成人血红蛋白(HbA)是由两条α‑珠蛋白链和两条β‑珠蛋白链组成的四聚体，其亚基构成为α₂β₂。每条珠蛋白链都折叠成特定的三级结构，其中含有一个形状精确的疏水口袋，用于容纳血红素基团。四条亚基通过非共价作用（主要是氢键、疏水接触和少量离子键）结合在一起，形成一个紧凑的、大致呈球形的四级结构。
The functional unit of haemoglobin is the αβ dimer; two such dimers associate to form the complete tetramer. During the transition between the oxygenated and deoxygenated states, the two αβ dimers slide and rotate relative to each other. This structural flexibility within the quaternary interface is the physical basis for cooperative oxygen binding and allosteric regulation.
血红蛋白的功能单元是αβ二聚体；两个这样的二聚体结合形成完整的四聚体。在氧合态与脱氧态之间的转变过程中，两个αβ二聚体彼此滑动并旋转。这种四级结构界面的结构柔性是协同氧结合和变构调节的物理基础。
3. The Haem Group and Iron Coordination | 血红素基团与铁配位
The haem prosthetic group is a protoporphyrin IX ring that tightly coordinates a single Fe²⁺ ion at its centre. The iron atom forms six coordination bonds: four bonds with the nitrogen atoms of the porphyrin ring, one bond with the imidazole side chain of a conserved proximal histidine residue (His F8) of the globin chain, and a sixth, vacant coordination site that can reversibly bind an O₂ molecule. A distal histidine (His E7) is located near the binding site and sterically restricts access, ensuring that the O₂ molecule binds in an end‑on geometry and reducing the risk of permanent oxidation of Fe²⁺ to Fe³⁺, which would generate methaemoglobin incapable of oxygen transport.
血红素辅基是一个原卟啉IX环，其中心紧密结合一个Fe²⁺离子。铁原子形成六根配位键：四根与卟啉环的氮原子结合，一根与珠蛋白链上一个保守的近端组氨酸残基(His F8)的咪唑侧链结合，第六个空置的配位位置则可逆地结合氧气分子。一个远端组氨酸(His E7)位于结合位点附近，空间上限制其可及性，确保O₂分子以端基方式结合，并降低Fe²⁺不可逆氧化为Fe³⁺（产生无法运输氧的高铁血红蛋白）的风险。
In deoxyhaemoglobin, the iron atom lies approximately 0.4 Å out of the porphyrin plane because the sixth coordination site is empty and the Fe²⁺ is in a slightly high‑spin state with a larger ionic radius. When O₂ binds, the iron moves into the plane of the ring, pulling the proximal histidine with it. This subtle but crucial conformational change is transmitted through the globin chain to the subunit interfaces, initiating the switch from the low‑affinity T (tense) state to the high‑affinity R (relaxed) state.
在脱氧血红蛋白中，由于第六个配位位置空缺，且Fe²⁺处于离子半径稍大的高自旋态，铁原子位于卟啉环平面外约0.4 Å处。当O₂结合时，铁原子移入环平面，并拉动近端组氨酸。这一细微但至关重要的构象变化通过珠蛋白链传递至亚基界面，从而启动从低亲和力的T态（紧张态）向高亲和力的R态（松弛态）的转换。
4. Oxygen Binding and Conformational Shift | 氧结合与构象转变
Deoxyhaemoglobin is stabilised in the T‑state by a network of salt bridges and hydrogen bonds at the αβ‑interface and between the two αβ dimers. When the first O₂ molecule binds to one subunit, the movement of the iron into the porphyrin plane triggers a local rearrangement that disrupts some of these stabilising interactions. This lowers the energy barrier for the remaining subunits to adopt the R‑state, making it easier for the next O₂ molecules to bind — the molecular explanation for positive cooperativity.
脱氧血红蛋白由αβ界面处以及两个αβ二聚体之间的盐桥和氢键网络稳定于T态。当第一个O₂分子结合到一个亚基上时，铁移入卟啉环平面引发的局部重排会破坏其中一些稳定作用。这降低了其余亚基转变为R态的能量壁垒，使后续O₂分子更容易结合——这正是正协同效应的分子解释。
The overall T‑to‑R quaternary transition involves a rotation of one αβ dimer relative to the other by about 15° and a narrowing of the central cavity. These large‑scale movements alter the environment around the haem groups and are the structural foundation of the Bohr effect and the regulation by 2,3‑bisphosphoglycerate. As a result, haemoglobin can switch rapidly between a tense, low‑affinity conformation in the tissues and a relaxed, high‑affinity conformation in the lungs.
整体的T态向R态四级结构转变涉及一个αβ二聚体相对于另一个约15°的旋转以及中央空腔的变窄。这些大规模的运动改变了血红素基团周围的环境，是波尔效应和2,3‑二磷酸甘油酸调节的结构基础。因此，血红蛋白能够在组织中的紧张低亲和力构象与肺部中的松弛高亲和力构象之间快速切换。
5. Cooperative Binding and Sigmoidal Kinetics | 协同结合与S形动力学
Because of positive cooperativity, the oxygen‑binding curve of haemoglobin has a sigmoidal (S‑shaped) profile when percentage saturation is plotted against pO₂. At very low pO₂ values, the protein is largely in the T‑state and binding is comparatively weak. Once the first O₂ is bound and the transition towards the R‑state has begun, the affinity for subsequent O₂ molecules increases markedly, creating a steep rise in the middle of the curve. The steep portion corresponds to the physiological pO₂ range encountered in tissues (approx. 2–6 kPa), where a small drop in oxygen tension triggers the release of a large amount of O₂.
由于正协同作用，当以氧饱和度百分比对pO₂作图时，血红蛋白的氧结合曲线呈S形（S形）。在非常低的pO₂值下，蛋白主要处于T态，结合相对较弱。一旦第一个O₂结合并且向R态的转变已经开始，对后续O₂分子的亲和力便显著增加，形成曲线中部的陡峭上升。该陡峭段对应组织中遇到的生理pO₂范围（约2–6 kPa），氧压的微小下降即可触发大量O₂的释放。
The degree of cooperativity can be quantified by the Hill coefficient (nH). For non‑ c. For haemoglobin, nH approaches 3, indicating significant positive cooperativity. The sigmoidal shape is therefore an elegant adaptation that enables haemoglobin to serve as an efficient oxygen deliverer, matching O₂ supply with metabolic demand.
协同作用的程度可用希尔系数(nH)定量。对于非协同结合（如肌红蛋白），nH = 1，曲线为双曲线。对血红蛋白而言，nH接近3，表明存在显著的正协同效应。因此，S形曲线是一种精巧的适应，使血红蛋白能够充当高效的氧递送分子，使氧气供应与代谢需求相匹配。
Hb + 4O₂ ⇌ Hb(O₂)₄
（此平衡极大地受构象状态和别构效应物的影响）
6. The Oxygen Dissociation Curve | 氧解离曲线
The oxygen dissociation curve relates the percentage saturation of haemoglobin with oxygen to the pO₂. A key quantitative parameter is p50, the pO₂ at which haemoglobin is 50% saturated. For normal adult haemoglobin at pH 7.4 and 37 °C, p50 is approximately 3.5 kPa (26 mmHg). A leftward shift of the curve means that p50 decreases, reflecting a higher oxygen affinity; a rightward shift corresponds to a larger p50 and a lower affinity.
氧解离曲线反映了血红蛋白氧饱和度百分数与pO₂之间的关系。一个关键的定量参数是p50，即血红蛋白达到50%饱和时的pO₂。对于正常成人血红蛋白（pH 7.4，37 °C），p50约为3.5 kPa (26 mmHg)。曲线左移意味着p50下降，反映氧亲和力升高；右移则对应p50增大和亲和力降低。
In the pulmonary capillaries, where pO₂ is about 13.3 kPa (100 mmHg), haemoglobin becomes approximately 97–98% saturated. In resting systemic tissues, with a pO₂ of around 5.3 kPa (40 mmHg), saturation falls to roughly 75%, indicating that about 22–25% of the bound O₂ is released. During vigorous exercise, tissue pO₂ can drop further, pushing the saturation point deeper into the steepest part of the curve and enabling a significantly larger fraction of O₂ to be offloaded.
在肺毛细血管中，pO₂约为13.3 kPa (100 mmHg)，血红蛋白约达97–98%饱和。在静息的全身组织中，pO₂约为5.3 kPa (40 mmHg)，饱和度降至约75%，表明约有22–25%的结合O₂被释放。在剧烈运动时，组织pO₂可进一步下降，将饱和点推入曲线的最陡峭部分，从而显著增加O₂的释放比例。
7. The Bohr Effect: pH and CO₂ | 波尔效应：pH与二氧化碳
The Bohr effect is the phenomenon whereby increasing concentrations of H⁺ (lower pH) or CO₂ reduce the oxygen affinity of haemoglobin, shifting the dissociation curve to the right. Actively metabolising tissues produce CO₂ and lactic acid, both of which lower the local pH. The resulting rightward shift enhances O₂ unloading precisely in areas where metabolic demand is high. In the lungs, where CO₂ is exhaled and pH rises, the curve shifts back to the left, promoting O₂ loading.
波尔效应是指H⁺浓度升高（pH降低）或CO₂浓度增加导致血红蛋白氧亲和力下降，使解离曲线右移的现象。代谢活跃的组织产生CO₂和乳酸，两者均降低局部pH。由此产生的右移在代谢需求高的区域增强了O₂的释放。在肺部，由于CO₂被呼出且pH升高，曲线回移向左，促进O₂装载。
At the molecular level, H⁺ ions bind to ionisable side chains (particularly histidine residues) in the globin chains, forming additional salt bridges that stabilise the T‑state. Likewise, CO₂ can react with the N‑terminal amino groups of both α and β chains to form carbamino compounds, which also stabilise the deoxygenated conformation. Consequently, tissues with high CO₂ output and high H⁺ production receive a greater supply of oxygen from the same amount of haemoglobin.
在分子水平上，H⁺与珠蛋白链中可离子化的侧链（特别是组氨酸残基）结合，形成额外的盐桥，稳定T态。同时，CO₂可与α链和β链的N端氨基反应形成氨基甲酰化合物，这也稳定了脱氧构象。因此，CO₂产出和H⁺生成量高的组织从同等量的血红蛋白中获得更多的氧气供应。
| Factor | Effect on curve | Mechanism |
| ↑ H⁺ (↓ pH) | Right shift (↓ affinity) | Stabilises T‑state via salt bridges |
| ↑ CO₂ | Right shift | Carbamino formation stabilises T‑state; also ↓ pH via carbonic acid |
