Histol Histopath (1989) 4: 509-51 4 Histology and Histopathology

Invited Re vie W

The phylogenetic odyssey of the erythrocyte. I : the universal respiratory pigment

Chester A. Glomski and Judith Tamburlin Department of Anatomical Sciences, State University of New York at Buffalo, New York, USA

Summary. Hemoglobin is a molecular entity that is Discussion capable of reversibly binding and releasing oxygen in either extra- or intracellular milieus. It is present in The hemoglobin molecule is truly ancient and has scattered invertebrates in physical solution or in cellular been extant for millenia. It has a long evolutionary sites while in it is universally located in history and in fact can be viewed as a molecular clock. It circulating erythrocytes. These cells serve as the vehicle has been used to date the separation of the vertebrates for and otherwise foster the optimum utilization hemo- and invertebrates some 1000 million years ago. The . Hernoglobin's variable sphere of respiratory emergence of a hemoglobin molecule with activities can be viewed as reflecting the specific differing alpha and beta globin chains occurred 500-600 requirements for each organism in which it is observed. million years later. Hemoglobin has persisted since those Once these concepts have been established and the primordial events and has become established as the advantages and limitations of its cytologic packaging single, dominant, essentially universal respiratory recognized, the study of the erythrocyte as expressed in pigment of the vertebrates. By definition respiratory its dimensions, colligative aspects, geometry, internal pigments, of which hemoglobin is the prime represen- morphology and pathologic variations can be approached tative, are inherently pigmented metallo- having in a purposeful manner. the unique ability of reversible binding with oxygen. Although other phylogenetically transitory respiratory Key words: Hemoglobin, Erythrocyte, Comparative molecules have appeared in the invertebrates (e.g. hematology, Respiratory pigments , chlorocruorin) none have approached the importance of hernoglobin. It has become increasingly apparent that hemoglobin enjoys a remarkably broad Introduction biological distribution. It is identifiable in some bacteria, occasional molds, some single celled animals (e.g. the The erythrocyte, by far the most numerous cell in the ciliated protozoans Paramecium caudatum and circulating , is the major carrier of oxygen in man Tetrahymena pyriformis) , scattered invertebrates, and all other vertebrates. This cell is uniquely adapted essentially all vertebrates and most surprisingly in the for this function by its high content of an iron-bearing, plant kingdom. This molecule has been identified in the oxygen binding , hemoglobin. An understanding nitrogen fixing nodules on the roots of some legumunous of the red cell's functional and pathologic activities in plants as soy bean plants and alfalfa as well as in the non- man (as well as in other vertebrates) can be approached nodulating plant Trema tomentosa. Indeed, recent by first assessing the phylogenetic expression and isolation of the hemoglobin gene from plant root cells has variation in activity of its respiratory pigment. This suggested that hemoglobin genes, inherited from an establishes a basis for an enhanced, continued study and ancestor common to plants and animals, might be deeper understanding of this cell. Hemoglobin is central present in all plants. All aerobic organisms are capable of to the biology of the erythrocyte. synthesizing catalysts such as the -containing . Consequently they can be viewed as potential carriers of the heme moiety of hemoglobin. Offprint requests to: Dr. Chester A. Glomski, M.D., Ph.D., Department The limiting factor in the appearance of hemoglobin of Anatomical Sciences, State University of New York at Buffalo, apparently lies in the evolved ability to synthesize the Buffalo, New York 14214, U.S.A. highly specific proteins () which when linked with Erythrocyte odyssey

into a tetramer composed of two alpha and beta protein units. Thi\ developmental process is believed to have occurred after the evolution ol the jawless fish (e.g. lamprey ancl hagfish) which do not demonstratc heterogeneous composecl of pairs of differing globin chains but before the cartilaginous fish (sharks) and all other vertebrates which do manifest this progression. The evolutionary ascendancc to polymeric hemoglobins can bc appreciated when viewed in light of Alfalfa, a the benefits that are obtainable by legumumus polymeric as opposed to monomeric plant forms. The oxygenation of one heme in an oligomeric hemoglobin molecule, for example, subsequently permits an adjacent fellow heme to bind oxygen with Partial Pressure 0, (mm Hg) greater facility. This alteration in binding affinity, 1 termed heme-heme interaction Fig. 1. Typ~cal s~gmo~dhemoglob~n oxygen d~ssoc~at~oncurve or cooperativity, is due to small (human hernoglobin). The Bohr effect is also ~llustrated.Note the changes in the three dimensional progressive shift of the dissociation curve to the right with a structure of the molecule. An decrease in pH. After VanSlyke and Peters. extension of this enhancement is the observation that all heme establish the constraints of its remarkable property hemoglobin molecules in a of reversible binding and release of oxygen. solution or within an erythrocyte The simplest molecular configuration of hemoglobin become equally oxygenated consists of one heme prosthetic group (a porphyrin ring when exposed to a given volume complexed with a single atom of iron) attached to one of oxygen; e.g. if a solution of globin chain approximately 150 amino acids in length. human (tetrameric) hemoglobin The molecular weight is approximately 17,000 daltons. is 50% saturated with oxygen all Hemoglobins of this order can be found in the free of the molecules will have two coelomocytes of some (multisegmented Representative oxygenated ferrous atoms. This worms), the larva of the insect Chironomous and in some (multisegmented characteristic also leads to sea cucumbers. The molecular weights of the primitive marine worm) the establishment of the well (invertebrate) hemoglobins that exist free in solution recognized sigmoid hemoglobin oxygen dissociation conversely, tend to be markedly greater and frequently curve which is ideally suited for taking up and release of approach three million daltons as a result of a high oxygen under different conditions (Fig. 1). Monomeric degree of polymerization. Their large molecular dimen- hemoglobins, in contradistinction, usually have hyperbolic sions serve to prevent them from diffusing out of the dissociation curves. The heme moiety of the molecule plasma into the tissues. Most intracellular invertebrate can be considered evolutionarily stable and responsible hemoglobins are monomers or dimers of the basic for the reversible binding of oxygen while the globin is molecular model although tetramers and larger forms structurally variable and through this variation determines can be represented. The characteristic the conditions under which oxygen binding and release tetrameric molecule of vertebrates will be favored (CO,, temperature, oxygen tension, (including man) most likely arose by organic phosphate and inorganic ion levels etc.). the joining of two dimers. The Hemoglobin molecules can therefore be viewed as vertebrates, with the exception of the cctailor made,, for specific species and temporal jawless fish (Agnatha. cyclostomes), all requisites. Man himself demonstrates at least four have a more complex, tetrameric different hemoglobins hemoglobin containing two pairs of during his existence. dissimilar (alpha and beta) globin the Gower I and 'k&~??%wd* chains; it has an average molecular I1 hemoglobins of .&+;4$$:$$;ifGf;ii%a$ weight of 68,000. It has been concluded ;4(r early embryonic life. ' .&%&2"@-4.,> that the original primitive vertebrate fetal and adult Parameoum hemoglobin consisted of four heme hemoglobins. Thc caudatum, a units and four identical polypeptide bRepresentative Sea Cucumber, chicken protozoan chains; it evolved by gene duplication multiple hemoglobins a holothurian Erythrocyte odyssey

during its lifetime (hemoglobins P, E, D, A and H) while interpreted as the net result of its fetal and adult hemoglobins are commonplace among two major functions, the loading mammalians. The maturation of a tadpole to a frog is and unloading of oxygen. The similarly associated with a molecular change of its effects of ambient organismnl hemoglobin. Some fish concomitantly manifest two conditions upon hemoglobin at the types of hemoglobin, one highly sensitive to acid pH and oxygen-rich side of the cyelc j one that is acid-insensitive. Certain representatives of (interface with air or water at the, the annelids and molluscs (bivalves or clams) that have gills, lungs, etc.) and the physiologic erythrocytes demonstrate the simultaneous existence of circumstances at the oxygen-pool- mono- and polymeric intracellular hemoglobins. The (tissue) side have to be both taken arcid clam Noetia ponderosa (a colloquial .blood clamn) into balanced consideration. Thu\. possesses erythrocytes with two dimeric hemoglobins fish with both acid sensitive an(l with own ou\cen-binding properties. It can be acid independent hemoglobins (e.g. ~i'ilna, iiaina gi,~;,,,~ assumed that each rainbow trout, Salmo irideus,) are molecular version in said to benefit from their bimolecular profile during ,In organism reflects extreme swimming effort at which time significant certain characteristics amounts of lactic acid are likely to be accumulated. The ,~ndbiochemical biases resultant drop in blood pH would prevent an acid (not always apparent) sensitive (Bohr effect) hemoglobin from combining with Mako or Sharp Nose Makerel that are desired at a any oxygen and lead to asphyxia. The presence of an acid Shark, lsurus oxyrinchus given period of life or independent hemoglobin however, insures that some under certain activity. hemoglobin can bind with oxygen even during maximal The oxygen dissociation curves of the hemoglobin in activity. various animals sometimes reveal these preferred There is no smooth evolutionary expression of features. For example, the llama which inhabits the hemoglobin as it progresses from the free, extracellular terrain of the Andes mountains has a dissociation curve to the cellular milieu or from invertebrate to vertebrate that reveals a marked avidity (loading capacity) for species. The blood or oxygen at the lowest of oxygen concentrations. This is of hemolymph of invertebrates obvious benefit to an animal that must capture its 15characterized by respiratory required oxygen in a rarefied atmosphere. Another (frequently other useful feature of most hemoglobins is that at lower pH than hemoglobin) existing levels their dissociation curve shifts to the right thereby free in the plasma in contrast facilitating the release of oxygen. This favorable to the vertebrates which characteristic is manifested in venous blood which has have specialized, circulating. increased acidity due to its higher levels of carbon hemoglobin-containing cells dioxide. This phenomenon is known as the Bohr effect Mouse, MUS muscu/us (erythrocytes) in their vascu- (Fig. 1). The hemoglobins of small mammals demonstra- lature. It is possible and te a greater Bohr shift than those of the larger species, sometimes conceptually convenient, however, to i.e. the incremental volume of oxygen released per unit identify lower forms, so called ccspecies of hemoglobin increase in acidity will be proportionately greater transition.. In such cases this molecule is observed in in smaller versus larger animals. The Bohr effect is physical solution, stored in fixed, non-motile cells or consequently progres- exists simultaneously both in cells and in the free state. sively greater in Such models often yield unanticipated insight into elephants, cattle, hop. the modes of harnessing hemoglobin's respiratory man, rabbits and micc capability. In addition, because of the absence of a This is seen as a predictable, stepwise pattern in the phylogenetic reflection of and in Tadpole of Common Frog, expression of hemoglobin and its cytophores it is possible physiologic concert with Rana temporana to identify ccearly or delayed. appearances of circulating the metabolic rate which erythrocytes on either side of the boundary between the increases as the size of an animal diminishes. The result vertebrates and invertebrates. thereby achieved is an improved availability of oxygen Numerous polychaete annelids (multisegmented, for subjects with the greatest need. The Bohr effect is not bristly marine worms) such as the bloodworm Glycera manifested by monomeric hemeproteins. It is typically dibranchiata have ,. absent or not prominent in annelid erythrocytic or free hemoglobin in their hemoglobin and is likewise not expressed in some free coelomic cells. It submammalian vertebrates as frog tadpoles and certain is usually of modest salamanders, e.g. Cryptobranchus alleganiensis, the molecular weight and hellbender. may be mono-. di- The benefits andlor disadvantages of a given or polymeric. This molecular configuration of hemoglobin in an animal are evolutionary cellular Rainbow Trout, Salmo ~rndeus Erythrocyte odyssey

4 advance approaches- - represented in the invertebrates, the cephalopods the limits seen in considered to be the most advanced molluscs as well as the invertebrates. For this zenith of the evolution of invertebrates nevertheless fail to reason two genera in demonstrate erythrocytic and extracellular hemoglobins. I he annelid phylum, The molluscan erythrocytic hemoglobins not surprisingly Thalassema and Mage- are typically small (mono- to oligomeric), as seen in the Iona. both of which cockle Anadara ovalis (the blood ark). Conversely, the clemonstrate free cells Planorbis corneus (a European fresh water snail, the great Lvntaining a respiratory ~.;~rn\Ii(>rn)c\-Iiihits an intr;r\.nscular hemoglobin with a Theiepus cr~spus, pigment, are sometimes molecular weight in an annelidan worm cited as filling the L,\-cessof one million evolutionary gap bet- c'altons. Perhaps the ween dissolved, free and cellular packaged respiratory most prodigious inver- pigments. The organism Magelona papillicornis is tebrate or vertebrate additionally unique in that it has true erythroplastids hemoglobin is that (denucleated erythrocytes), its pigment however is observed in the clam which does contain iron but not a porphyrin Cardita borealis. Its ring. Telepus crispus and Pista pacifica, also marine molecular weight is in annelids, simultaneously demonstrate both extracellular the order of twelve vascular and intracellular coelomocytic hemoglobins. ,

numerous entomostracan crustaceans (a class of simple, brates to demonstrate small, aquatic with a chitinous exoskeleton, red cells. These e.g. Daphia magna, a water flea and Chirocephalus. An recognizably, poorly interesting respiratory phenomenon observed in the developed agnathic former species is the stimulation of the synthesis of species also posses5 hemoglobin in the absence of oxygen. the most primitive A small number of echinoderms (marine invertebrates hemoglobins observecl with a <>, e.g. sea urchins, starfish) utilize in vertebrates. The! hemoglobin, a fact recognized by the early hematologists reveal their incomplete \~chas Toll\ and Foett~ngerThe brick-red colored sea evolution bv a retention cucumber Cucumaria of monomkric hemo- Representat~veBr~ttle Star miniata has a modest globins. On the other an ech~noderm i\ number of hemoglo- hand the physiologic b~niferous cells in properties of their hemoglobins (especially the lamprey) ~tscoelomic fluid. do show some degree of molecular cooperativity; the Other members of the lamprey hemoglobin also has a Bohr effect while the class Holothuroidea hagfish does not. In some remarkable circumstances it is (sea cucumbers) with possible to identify a well developed vertebrate that has erythrocytes are Mol- evolved without the development of erythrocytes or padia arenicola, Caudina dissolved pigment in its plasma. This well-documented 6ot Fly and larva (bot or maggot), retetzii and Thyone. status is observed in certain antarctic ice fish (<>in this of specialized, hemoglobln species. Though devoid of hemoglobin they are said to bearing erythrocytes. The\c * have the enzymes related to carbon dioxide-bicarbon;~tc. cells are independent. W transport, another function of , mobile, deformable and water Flea, Daphn~aspec~es the red cell. Support for the 1 passive-flowing. In all but concept of temperature-assisted l? the mammals they can be expected to be oval in aqueous solubilization of oxygen configuration, biconvex, and permanently nucleated. is seen in another endemic fi' The amphioxus, (Branchiostoma lanceolatum), a Antarctic species, the Noto- ,I/ primitive, semitransparent fish-like animal that is theniidae. These teleostine recognized as an archetypal chordate (a cephalochordate) (bony) polar fish have low i.e. a species more taxanomically advanced than the hemoglobin levels, about 3-4 / ,S,,, invertebrates but still not attaining the developmental gmldl. This value is apparentl! stature of a vertebrate, has blood devoid of erythrocytes, within the physiologic limits ii' hemoglobin and other respiratory pigments. Other required for the organism due to Lamprey, primitive chordates (e.g. some urochordates) the significant level of oxygen Petrom~zonmarlnus have alternative non- dissolved in the blood. The larva hemoglobinous pig- of the eel (Anguilla anguilla) is another example of a ments in their circu- vertebrate that is adapted to exist without the benefit of lating cells. The erythrocytes or dissolved respiratory pigments. Rare ~'iwless fish (lamprey, instances have been recorded in which the African Petromyzon marinus clawed toad (Xenopus laevis), which normally has an dnd hagfish, Myxine erythrocyte count appropriate for an amphibian, was glutinosa are the found to have no erythrocytes in its blood. Whether this Cucumana mmlata, a Sea Cucumber most primitive verte- relates to a particular metabolic or pathologic state or is ~teodyssey

possible because of the toad's cutaneous respiration has Acknowledgements. The illustrations were rendered by Ms. Kathleen not been established. Long, medical illustrator and the typescript was prepared by Ms. The evolution of oxygen transport initiating with Theresa D'Angelo. The authors are greatly indebted to both simple physical aqueous solution, advancing to utilization individuals for their professional interest. Manuscript preparation was of an extracellular protein carrier (hemoglobin) and supported in part by Sub-Board I, State University of New York at terminating in erythrocytic microenvironmentalization Buffalo. of this respiratory pigment yielded ever increasingly efficient mechanisms of oxygenation required by complex, high energy dependent organisms. The development of the circulating red cell permits that Selected references attainment of higher concentrations of hemoglobin in the blood than would likely be physiologically feasible if it Bogusz D., Appleby C.A., Landemann J., Dennis E.S., Trinick M.J. were in free solution. Considering the fact that man's and Peacock W.J. (1988). Functioning haemoglobin genes in hemoglobin level is approximately 15g/100ml of blood, non-nodulating plants. Nature 331, 178-180. the absence of its erythrocellular vehicle would impose a Brunori M,, Giardina B., Bonaventura J., Barra D. and Antonini E. formidable solute load upon the plasma, far greater than (1973). Properties of fish hemoglobins. In: Comparative the aggregate of all other plasma proteins. Sea water, the Physiology. Bolis L., Schmidt-Nielsen K. and Maddrell, S.H.P. original milieu. has an oxygenic carrying capacity (eds). American Elsevier Publishing. New York. pp 447-492. of approximately 0.5cc/100ml while the addition of Ewer D.W. (1959). A toad (Xenopus laevis) without haemoglobin. mammalian hemoglobin to plasma increases this volume Nature 183,271. forty fold. It is noteworthy that hemoglobin's oxygen Foettinger A. (1880). Sur I'existence de I'hemoglobine chez les binding capability is greater in water than in the echinodermes. Arch. Biol. 1,405-41 3. mammalian erythrocyte because of the latter's ionic and Garlick R.L. and Terwilliger R.C. (1974). Coelomic cell hemoglobin organic phosphate constituents (e.g. diphosphoglycerate, of the terebillid polychaete, Thelepus crispus. Johnson. adenosine triphosphate). This diminution is recognized Structure and oxygen equilibrium. Comp. Biochem. Physiol. as a shift of the hemoglobin dissociation curve to the 478,543-552. right, i.e. a decrease in the oxygen affinity of the Hungerford H.B. (1982). Oxyhaemoglobinpresent in backswimmer hemoglobin from the maximum. The reduction from the Buenoa margaritacea bueno (hemiptera). Canad. Entomol. 54, theoretical potential can be viewed as a trade-off for the 262-263. physiologic~functionsperformed by these other hemato- Jolly J. (1923). TraiteTechnique D'Hematologie. Maloine A. etfils. genous components. Paris. pp 91 -1 03. The most important advantage of enclosing hemoglobin Keilin D. and Ryley J.F. (1953). Haemoglobin in protozoa. Nature in circulating cells is the opportunity to have different 172,451. chemical environments in the plasma and within the Nagel R.L. (1985). Molluscan hemoglobins. In: Blood Cells of erythrocyte. The intracellular pH of the erythrocyte is Marine Invertebrates: Experimental Systems in Cell Biology lower than that of plasma and both media have their own and Comparative Physiology. Cohen W.D. (ed). Alan R. Liss. individual molecular composition. Cellular packaging of New York. pp 228-247. hemoglobin allows its respiratory functions to be Prosser C.L. (1973). Respiratory functions of blood. In: Comparative promoted and shelters it from irreversible oxidation in Animal Physiology. 3rd ed. Prosser C.L. (ed). W.B. Saunders. the plasma by the development of appropriate protective Philadelphia. pp 31 7-360. intracellular enzymes. Its biological half-life is thus Runnegar B. (1982). A molecular-clock date for the origin of the extended by avoiding degradation in the plasma and also animal phyla. Lethaia 15, 199-205. by preventing its loss through phagocytic and renal Ruud J.T. (1958).Vertebrates without blood pigment: A study of the tubular activity. Mammalian hemoglobin free in the fish family Chaenichthyidae. Proc. 15th Int. Cong. Zool. 526-528. plasma in the concentration of 50mg/100ml solution, for Schmidt-Nielsen K. (1983). Blood. In: Animal ~hysiology: example, has a half-life of only about 40 minutes. The Adaptation and Environment. 3rd ed. Cambridge Univ. 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