Structure and function of haemoglobin

This chapter does not refer to any Section of the 2023 CICM Primary Syllabus, because nowhere in the Syllabus Document is there anything about the structure and function of haemoglobin. Foetal haemoglobin and dyshaemoglobins like methaemoglobin get their day in the sun, but the normal molecule is omitted. Fortunately, nobody ever looks at the Syllabus anyway, as assessment drives learning and the trainees have long ago realised that studying the previous exam papers is the only way to pass. In that shadow curriculum, there is a place for the discussion of haemoglobin, as it has come up several times in the previous papers:

  • Structure of haemoglobin:
    • Haemoglobin is a tetramer composed of four subunits, two α and two β 
    • These subunits form two identidal αβ dimers
    • Each subunit has a haem group
    • The quaternary structure of haemoglobin is essential for positive cooperativity:
      • Each time one of the monomers binds oxygen, the molecule of haemoglobin undergoes a conformational change
      • This changes the equlibrium constant for the next O2 molecule to bind the next subunit (increasing the affinity for O2)
      • This is described as a transition from the T ("tense") deoxygenated state to the R ("relaxed") oxygenated state, though a series of intermediate T-like and R-like states
      • This property is responsible for the sigmoid shape of the oxygen-haemoglobin dissociation curve
  • Haem groups are iron-containing molecules of protoporphyrin-IX with physiologically essential properties:
    • Hydrophilic cores and hydrophobic external chains, for positive cooperativity
    • Able to bind different gases (O2, NO, CO, H2S, etc)
    • Switching between Fe3+ and Fe2+ states allows it to participate in oxidation-reduction reactions
  • Function of haemoglobin
    • Oxygen transport
      • Increases the oxygen-carrying capacity of blood by ~ 50-100 times
      • Affinity for O2 is increased under conditions of high POand decreased under conditions of low PO2, enhancing the loading and unloading of oxygen
    • CO2 transport
      • 10-20% of total CO2 carriage in the blood as carbamino componds
      • Deoxygenated haemoglobin has a higher affinity for CO2 (Haldane effect)
    • Buffering
      • Deoxyhaemoglobin acts as a buffer (histidine residues with pKa = 6.8)
      • Of the plasma proteins, most of the buffering is done by haemoglobin, and it accounts for 50-60% of the total buffering capacity of the blood
    • Nitric oxide (NO) scavenging
      • NO binds to the ferrous (Fe2+) iron with great affinity; and this is the most physiologically important mechanism of limiting nitric oxide bioactivity
      • This plays a physiological role in the autoregulation of regional blood flow
      • Clinical implications and examples of this include:
        • Hypoxic pulmonary vasoconstriction,
        • Pulmonary hypertension seen with polycythaemia 
        • Sickle cell vasoconstrictive crisis
  • Metabolism of haemoglobin
    • Free haemoglobin is bound to haptoglobin and captured by macrophages
    • Free haem is bound to haemopexin and captured by macrophages
    • Haemoglobin in senescent erythrocytes is reclaimed by macrophages
    • Inside reticuloendothelial macrophages:
      • Globin molecules are catabolised and released as amino acids
      • Haem is stripped of iron and converted into biliverdin, then bilirubin
        • Bilirubin circulated bound to albumin and is conjugated in the liver
        • Conjugated bilirubin is screted and converted into urobilinogen in the bowel, then into stercobilinogen and then into stercobilin
        • Urobilinogen can also be reabsorbed and exreted in the urine as urobilin
      • Iron is stored in the reticuloendothelial cells as ferritin or exported into the circulation, in which case it circulates as transferrin
 

Being an important molecule, central to vertebrate life, it is not surprising that a lot is written about haemoglobin. For most normal people, the free articles by Thomas & Lumb (2012) or Marengo-Rowe (2006) will be enough. If for whatever reason a significantly larger block of text is required, Jensen et al (1998) have you covered, also gratis. And if some sort of breakdown overrides the reader's normal regulatory self-protective functions, "Hemoglobin" by Storz (2018) will be an excellent option, more like an extended fireside chat about protein research and evolution which keeps coming back to the topic of haemoglobin between sips of brandy. The tone of its chapter headings ("A study in scarlet", etc) and the occasional quote from Mephistopheles make it readily bingeable, and also should not deter the reader from regarding it as a serious reference text on the subject.

Chemistry of haemoglobin

This molecule represents the most populous faction of the bloodstream proteins. About 97% of the dry weight of red cells is all haemoglobin, and the average human circulatory system holds about 750g of this stuff (compare that to, for example, albumin, of which there is only 200g). You really could just describe the circulatory system as a large branching organ filled mainly with haemoglobin. The protein itself is about 64 kDa, and dissolves very well in water (for example, the contents of a red cell is an almost completely saturated solution of haemoglobin, at around 330g/L). 

It is not an especially stable molecule; or rather it performs poorly when stress-tested in conditions that lay outside of the normal physiological norms. It begins to denature if heated to 50º C, and by 65º C most of it becomes irreversibly destroyed (Rieder et al, 1970). If you drop the pH to 3.0 or below, it unfolds and the haem falls off. In case you want to play with some at home, powdered bovine haemoglobin is available (it has a good shelf life, and you reconstitute it as you want). It used to enrich agar,  create calibration solutions for measurement instruments, and create culinary abominations. Before you ask, yes acellular free haemoglobin solutions have also been considered as a resuscitation fluid, but unfortunately it is hideously toxic (more on that later).

Structure of haemoglobin

"The structure component was often only briefly described with a cursory overview provided; however, this component contributed around half of the available marks", complained the examiners in their comments for Question 11 from the first paper of 2020. Presumably, they wanted something more than "tetramer with haems". Haemoglobin structure is actually a rather deep rabbit hole, and the process of writing this section was supposed to be mainly an exercise in removing fascinating detail until something pragmatic and exam-focused remained. Yet, here we are.

Normally haemoglobin is depicted graphically as a ball of curled ribbons, illustrating that its secondary structure is a ball of α-helices. Alternatively, it can be depicted as a sort of blobogram, making it look more solid and molecule-like. Neither is of any use to the exam candidate, who cannot possibly be expected to draw the structure of haemoglobin in an exam. As it is never in the spirit of Φ to shy away from pointlessness, here they are:

pointless diagrams of the quaternary structure of haemoglobin

In case one may be interested in something less artificial, here a famous image from Longchamp et al (2016) illustrates a haemoglobin molecule alongside some of its peers (the images are somewhat uneven because the publisher decided to print them all at different scales, and they had to be resized).

single molecule protein images from Longchamp et al (2016)

So: what could you possibly be asked about the quaternary structure of haemoglobin? In their infinite cruelty, the examiners may be able to find some way of torturing the trainees with the structure-activity relationship. Specifically, they may latch onto the concept of positive cooperativity. In short:

  • Each of the four subunits bind oxygen independently.
  • Once an oxygen molecule is bound to one of the subunits, the oxygen affinity of the remaining subunits is increased.
  • This is because the binding of each oxygen molecule changes the shape of the tetramer, changing the equlibrium constant for the next O2 molecule to bind the next subunit more easily.
  • For example, when it has bound its full complement of oxygen molecules, one pair of αβ subunits in the fully oxygenated "relaxed" state appears rotated by 15° with respect to the other pair of subunits.
  • This conformational change produced by each subunit binding oxygen is described as a transition from the T ("tense") deoxygenated state to the R ("relaxed") oxygenated state. In this sense, oxygen is an allosteric modulator of the haemoglobin molecule.

Haem

Each haemoglobin monomer houses a haem group, and is structurally similar to myoglobin, which always exists as a monomer. Haem is a flat porphyrin ring, and the depiction of its molecular structure is as ubiquitous in textbooks as it is rare in exam papers, i.e. you'll never be asked to draw this on a piece of paper during a viva. Apart from demonstrating where the oxygen goes, this image really has no other redeeming qualities:

molecular structure of haem

For most normal people, the significance of this thing really will not become apparent from just a picture, and so a thousand words will have to do instead. In fact, what would be much better is sixteen pages of Prem Ponka (1999), explaining the wonders of haem. As virtually nobody will have the time to read this excellent paper, a summary of the main points follows:

In short what the hell is haem? If you were a chemist, you'd describe it as a complex of protoporphyrin IX and iron. Protoporphyrin IX is a porphyrin molecule, that is to say a macrocyclic ring formed of four smaller pyrrole rings, and it is number IX because Roman numerals are used to identify the positions of the methyl and vinyl groups on its periphery in the accepted system of porphyrin nomenclature (Moss et al, 1988). Protoporphyrin IX is in fact a rather ubiquitous molecule and is present in essentially all living things. Your sourdough experiment  uses it to measure oxygen tension. A similar structure with magnesium instead of iron in the centre is chlorophyll, and both are members of the metalloporphyrin family (researchers have managed to coerce other divalent metals in there, for example Junga et al (2010) forced it to bind copper and zinc in its cavity). Anyway,  when it binds iron specifically, you call it haem.

Haem, or heme with a different colonial history, is a rather special molecule, to the point where serious people write entire books to serenade it. It has multiple properties which make it incredibly versatile and biologically useful. For example, that iron bit in the centre is hydrophilic, but the vinyl and methyl groups on the outside are hydrophobic, which makes this molecule able to fit into protein structures in a variety of ways, for example into a hydrophobic pocket which can open and close as needed (this is the basis of the effect of positive cooperativity on oxygen binding).  Moreover, the iron ion can have six ligands, but in raw haem only has four coordinated ligands, which means it can bind two more: one is usually an imidazole side chain and the other is a gas molecule, which can be oxygen, carbon monoxide, nitric oxide, hydrogen sulfide, etc - in short it can act as a transporter for small molecules. On top of that, the heme iron ion can easily go back and forth between oxidation states: it can be ferric (Fe3+) or ferrous (Fe2+) and this gives it the ability to get involved in oxidation-reduction reactions. In the human organism, about 80% of the total haem is in haemoglobin, and most of the rest is in the liver, where it forms an integral part of the CYP450 enzyme system, not to mention electron transport chains, peroxidases, dehydrogenases, and numerous other enzymes.

Function of haemoglobin

Without going on an extensive tangent on something covered well enough in another section, the following is a summary of the functions of haemoglobin, with the headings highlighted by links to wherever more detail is available. Oxygen and carbon dioxide transport are fairly straightforward and there's plenty of material to read about it (here and elsewhere). 

  • Oxygen transport:
    • Haemoglobin increases the oxygen-carrying capacity of blood by 50-100 times
    • Its affinity for oxygen is described by the oxygen-haemoglobin dissociation curve, which has a sigmoid shape because:
      • Affinity for O2 is increased under conditions of high PO2, increasing its ability to collect oxygen from the lungs
      • Affinity for O2 is decreased under conditions of low PO2, enhancing the unloading of oxygen at the tissues
  • Carbon dioxide transport: 
    • Haemoglobin binds CO2 with a high affinity in its deoxygenated state  (the Haldane effect), and releases it when it becomes oxygenated and loses its affinity for CO2 (the reverse Haldane effect)
    • About 10-20% of the total carriage of CO2 in the blood is owing to this mechanism
  • Buffering:
    • Haemoglobin has many histidine residue groups with a protonation pKa close to 6.8
    • This allows it to act as a buffer at physiological pH, as these histidine molecules act as proton acceptors
    • This buffering capacity increases in its deoxygenated state.
    • As haemoglobin is one of the most populous proteins, it contributes a large proportion of the total buffering in the extracellular fluid, and this is incorporated into the calculation of the standard base excess.
    • The total buffering power of haemoglobin is thought to account for up to 50-60% of the total buffering capacity of the blood, the rest being attributable to the bicarbonate and other proteins. This figure is seen in many textbooks and fine publications (eg. Rubana & Aulik, 2009), and appears to be based on the work done by Ellison Straumfjord and Hummel (1958), who compared the buffering capacity of whole blood to that of acellular plasma (38.5 mEq/L/pH, whereas for plasma alone it was 16.1 mEq/L/pH).
  • Nitric oxide scavenging:
    • Nitric oxide binds to the ferrous (Fe2+) iron with great affinity, creating nitrosylhaemoglobin
    • This reaction is rate-limited by the fact that haemoglobin is packaged in erythrocytes, which increases the useful half-life of nitric oxide.
    • By removing nitric oxide from the peripheral circulation, haemoglobin contributes to the regional autoregulation of blood flow
    • This mechanism is the most physiologically important mechanism of limiting nitric oxide bioactivity
    • Clinical implications and examples of this include:

Nitric oxide scavenger role of haemoglobin

Nitric oxide (NO) is a free radical mediator of vasodilation, used for that purpose medically as an inhaled pulmonary vasodilator. It happens to have an extremely high affinity for haemoglobin, where it rapidly binds to the Fe2+ (ferrous) iron and forms nitrosylhaemoglobin. The affinity between NO and haemoglobin is so great that the reaction between deoxygenated haemoglobin and nitric oxide is only rate-limited by its diffusion into the haem pocket. i.e. with enough haemoglobin all nitric oxide could be removed from a solution within moments.

Specifically, the normal half-life of nitric oxide in acellular plasma is about 1 minute, but  Helms & Kim-Shapiro (2014) calculated that with a haemoglobin concentration of 10 mmol/L (about 64g/L) the half-life of nitric oxide in solution would be down to about 1 µs.  In other words, there would be absolutely no way for nitric oxide to ever act as an endothelium-derived relaxation factor.  Let's say an amount of NO  gets formed at the endothelial surface and is immediately exposed to haemoglobin. Over that 1 µs half-life, a molecule of NO could only diffuse about 0.1 µm from the site of its synthesis. It would never reach the vascular smooth muscle or really anything else of any interest.

This nitric oxide scavenger role of free acellular haemoglobin is the specific thing that has made it unattractive as a resuscitation fluid, to the point where free haemoglobin solution experiments in animals have been so discouraging that there does not appear to have ever been any human trials.  Nitric oxide is clearly an essential part of regional blood flow autoregulation and to remove it from the endothelium on a whole-body scale will result in massive and widespread vasoconstriction. De Figueiredo et al (1997) gave about 200ml of a 100g/L solution to some severely shocked pigs and determined that, though blood pressure improved, the cardiac output did not, and the peripheral and pulmonary arterial pressure increased markedly (the latter almost tripled). 

In short, the only reason that nitric oxide can function as a vasodilator is because haemoglobin is packaged inside red cells, which increases the diffusion distance for NO molecules and allows it to have a longer half-life (Lancaster et al, 1994). Thus, haemoglobin, by acting as a regulator of regional nitic oxide content in blood, contributes to the autoregulation of regional blood flow. Specifically, it is the mechanism of making sure that the vasodilator effects of nitric oxide remain in check. Several other mechanisms exist, which could be mentioned (Gladwin et al, 2004, give a detailed overview)- in summary, haemoglobin also controls the conversion and reverse-conversion of nitric oxide to and from nitrite (NO2-), which behaves as something of a nitric oxide pool. The direction of these reactions depends on the oxygenation state of haemoglobin and results in increased nitric oxide availability in the presence of deoxyhaemoglobin, or its decreased availability in the presence of oxyhaemoglobin, which is one of the mechanisms proposed to explain hypoxia-mediated regional vasodilation in the peripheral circulation and hypoxic pulmonary vasoconstriction in the pulmonary circulation.

Metabolic fate of haemoglobin

Question 15 from the first paper of 2012 specifically focused 60% of the mark on the breakdown of haemoglobin. "Globin (protein), Fe and haem (porphyrin ring) were all expected to be considered separately", the examiners added in their comments, though this would not be obvious from the actual question they asked, which may be why "many candidates omitted one or all". The pass rate was still 60%, suggesting the majority of us scraped through by their thorough understanding of RBC production (the other 40% of the marks). Anyway: the intention of this aside was to point out that the discussion of haemoglobin metabolism needs to take all of these elements separately. Additionally, to be a completely detestable pedant, one should separate things into the metabolic fate of conventionally metabolised well-behaved haemoglobin being reclaimed from senescent red cells, and wild free acellular haemoglobin released in the course of unplanned haemolysis.

Metabolic fate of free acellular haemoglobin

As mentioned above, free haemoglobin in the circulation is a hideous enemy and must be neutralised. Several mechanisms exist to make this happen, described beautifully by Schaer et al (2013). To summarise, in the inhospitalble environment of the plasma, a fair proportion of the inherently fragile haemoglobin molecules fall apart into αβ heterodimers. This is even worse, as they are smaller (32 kDa) and therefore able to penetrate into all sorts of delicate places, for example the renal tubule and the endothelial glycocalyx. The main way of dealing with these dissolved molecules is through soluble scavenger proteins which can bind and detoxify them. The most important of these are haptoglobin and haemopexin.

Haptoglobin is a huge protein which forms an even huger complex with haemoglobin (150 kDa), functionally limiting its penetration into vulnerable sites and making it less available for criminal nitric oxide scavenging activity. Outside of wartime, this step also prevents valuable iron from escaping into the urine, and allows it to be reclaimed.  Haptoglobin-haemoglobin complexes can realistically continue to circulate harmlessly for a while, but in reality they are quickly snaffled up by reticuloendothelial macrophages, which have high-affinity receptors for the complex (HbSR/CD163). The affinity is so high that any substantial haemolysis rapidly depletes the serum levels of circulating haptoglobin, making haptoglobin an effective biomarker to detect the aforementioned haemolysis.  The receptor-ligand complex is then endocytosed and committed to the sort of phagolysosome that is the final common pathway for all haemoglobin (Madsen et al, 2001).

Haemopexin is the less-spoken of haemin-binding β-glycoprotein. During haemolysis, some of the released haemoglobin will break down, releasing haem and haemin (hemin?), which is what you call ferric haem with an oxidised Fe3+ ion. These molecules have unpleasant lipid and protein-modifying effects, and it is in your interest to get rid of it. Haemopexin scavenges them and allows them to become endocytosed and degraded by hepatocytes (Morgan, 1976)

Fate of erythrocytes and the haemoglobin within them

Without recapitulating the content of other chapters, a normal red blood cell usually has a lifespan of about 120 days, during which, like a man, it becomes increasingly inflexible and physically abnormal. The ultimate destination for such cells is the reticuloendothelial system (particularly the spleen). There, cells that are insufficiently deformable to negotiate tight sinusoids are trapped and destroyed by reticuloendothelial macrophages (Huijes et al, 2018). This destruction is a nonviolent and totally consensual vore process, where a macrophage engulfs an erythrocyte and carefully dismantles it within a phagolysosome (Mebius & Kraal, 2005). Here, an amazing SEM image from Sasaki et al (2009) demonstrates several smooth erythrocytes bound membrane-to-membrane to a macrophage which appears to be drinking them gently:

erythrophagolysosome from Sasaki et al, 2009

These macrophages, or specifically the phagolysosome, are also the site of the rest of the steps which ultimately liberate the constituent parts of metabolised haemoglobin back into the circulation. Basically all of haemoglobin metabolism takes place here, and only the metabolic endproducts (bilirubin, iron, amino acids) are exported by these cells.

Metabolic fate of the haemoglobin protein in macrophages

As mentioned, all of the major pathways of haemoglobin metabolism lead to the macrophage  phagolysosome (though hepatocytes can also endocytose haem-haemopexin complexes). The metabolism of the globin element is relatively unexciting and resembles the metabolism of other ingested proteins, or at least so we think. For something stated with such certainty in the literature, we really have very little to back the assertion that the globin molecule is broken down into constituent amino acids and released into the circulation for recycling (even though this is the most logical outcome). The only series of experiments that point to this conclusion were performed by Ehrenreich & Cohn (1968), who fed radiolabeled haemoglobin to some rat macrophages. Much of the radioactive isotope was subsequently recovered from the solution which the cells were bathed in, in the form of labelled amino acids. The macrophages seemed to just extrude them when they were finished metabolising the proteins. "The present results show that by pinocytosis and subsequent digestion processes macrophages are capable of producing utilizable amino acids from ambient proteins", the investigators concluded. Something very similar seems to happen to albumin, which also finds its end in the reticuloendothelial system, and which is also degraded back into amino acids and peptide fragments.  

Anyway: the globin component is boring. The most interesting part of haemoglobin metabolism is what happens to haem.

Metabolic fate of haem

The lysosomal breakdown of haemoglobin monomers releases the haem molecule. Right there in the macrophage, it is degraded into its constituent parts, liberating iron for reuse by the organism. One possible way of presenting this pathway is by a flow diagram:

Pathway of haem metabolism

Or, in the form of words:

  • Haem oxygenase selectively cleaves the α-methene bridge of the porphyrin ring and "unrolls" it in the presence of oxygen, generating biliverdin - the blue-green pigment responsible for the awesome appearance of bruises. 
  • Iron is released by this first step. It also releases carbon monoxide, and is responsible for the normal low level of carbon monoxide seen on every blood gas.
  • Biliverdin is reasonably water-soluble and could possibly just be cleared directly, but in mammals it gets converted into bilirubin by biliverdin reductase
  • Bilirubin is lipophilic, highly protein-bound, and circulates attached to albumin. 
  • In the liver, bilirubin is conjugated with glucouronide to make it water-soluble, and the resulting diglucouronide is eliminated via the bile
  • The reduction of bilirubin by intestinal bacteria produces urobilinogen
  • Urobilinogen is reduced directly to stercobilinogen and then oxidised to stercobilin, which gives faeces their colour.
  • Urobilinogen can also be reabsorbed from the gut, and small amounts of it are excreted in the urine as urobilin

In all honesty the last few steps are probably immaterial, as they do not contribute overmuch to the iron cycle, but it appears that they should be mentioned in an answer about haemoglobin metabolism, as examiner comments mention it. One could also go on about the fates of urobilinogen and stercobilinogen 

Metabolic fate of iron

Following the breakdown of globin and haem, the macrophages will be full of iron. In fact hepatocytes and macrophages are one of the most important stores of body iron,  stored inside them as ferritin (Hentze et a, 2010). It can also be exported from these macrophages by the actions of  Cp is commonly present on macrophage membranes and is thought to mediate iron oxidation to facilitate its export out of the cell by ferroportin-1 (a membrane transporter) and ceruloplasmin (thought to mediate iron oxidation, which is an essential step in getting it out of the cell). It can then circulate around in a complex with transferrin, and do a whole host of fascinating things, but to go into any more detail about the comings and goings of iron would be better left to a whole separate chapter dedicated to its metabolism.

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