The Bohr effect before Perutz.

Before the outbreak of World War II, Jeffries Wyman postulated that the Bohr effect in hemoglobin demanded the oxygen linked dissociation of the imidazole of two histidines of the polypeptide. This proposal emerged from a rigorous analysis of the acid-base titration curves of oxy- and deoxy-hemoglobin, at a time when the information on the chemistry and structure of the protein was essentially nil. The magnetochemical properties of hemoglobin led Linus Pauling to hypothesize that the (so called) Bohr histidines were coordinated to the heme iron in the fifth and sixth positions; and Wyman shared this opinion. However, this structural hypothesis was abandoned in 1951 when J. Wyman and D. W. Allen proposed the pK shift of the oxygen linked histidines to be the result of "...a change of configuration of the hemoglobin molecule as a whole accompanying oxygenation." This shift in paradigm, that was published well before the 3D structure of hemoglobin was solved by M.F. Perutz, paved the way to the concept of allostery. After 1960 the availability of the crystallographic structure opened new horizons to the interpretation of the allosteric properties of hemoglobin.

Milestones in the history of hemoglobin research (in memory of professor Titus H.J. Huisman).

Professor Titus H.J. Huisman is best known for his work on hemoglobin (Hb) variants. To date, more than 1,000 Hb variants have been discovered and characterized, of which about one-third were discovered in Titus Huisman's laboratory at the Medical College of Georgia, Augusta, GA, USA. A registry of these Hb variants and other information, a legacy from Professor Huisman, is now available online, at HbVar database (hhtp://globin.bx.psu.edu/hbvar). During the last century, major developments in Hb research have been made using physical, chemical, physiological and genetic methods. This review highlights the milestones and key developments in Hb research most relevant to hematologists, and that have impacted our understanding and management of the thalassemias and sickle cell disease.

The classic: The chemical constitution of respiration ferment.

This Classic Article is a reprint of the original work by Otto Heinrich Warburg, The Chemical Constitution of Respiration Ferment. An accompanying biographical sketch of Otto Heinrich Warburg, PhD, MD, is available at DOI 10.1007/s11999-010-1533-z . The Classic Article is from Warburg O. The chemical constitution of respiration ferment. Science. 1928;68:437-443. Reprinted with permission from AAAS.

Chapter 1: building the ground for the first two protein structures: myoglobin and haemoglobin.

Fifty years ago, Max Perutz and John Kendrew at Cambridge University achieved something that many people at the time considered impossible: they were the first to use x-ray crystallography to decipher the molecular structures of proteins: haemoglobin and myoglobin. They found that both molecules were built from Linus Pauling's alpha helices, but folded and packed together in a complicated manner that never could have been deciphered by any other technique.With structure information in hand they could then explain how haemoglobin in the bloodstream binds and releases oxygen on cue, how it passes its cargo on to the related storage protein myoglobin, and how a single amino acid mutation can produce the catastrophe known as sickle-cell anemia. Perutz and Kendrew also observed that the folding of helices was identical in myoglobin and the two chains of haemoglobin, and this along with the simultaneously evolving new technique of amino acid sequence analysis established for the first time the concept of molecular evolution. The crystallographic puzzle was "cracked" by Perutz when he demonstrated that the binding of only two heavy metal atoms to horse haemoglobin changed the x-ray pattern enough to allow him to solve the "phase problem" and circumvent the main obstacle to protein crystal structure analysis. Because myoglobin has a single chain whereas haemoglobin has four, Kendrew's work with myoglobin progressed more rapidly; a low resolution structure appeared in 1956 and the high resolution structure in 1959. That same year saw the low resolution picture of haemoglobin, and the high resolution structure followed shortly thereafter. Much of the work in structure analysis was carried out by visiting postdoctoral fellows and technicians, under the watchful eye of Perutz and Kendrew. This celebratory review has been written by three of those former postdoctorals: Strandberg and Dickerson from the myoglobin project, and Rossmann from the haemoglobin.

Hemoglobin research and the origins of molecular medicine.

Much of our understanding of human physiology, and of many aspects of pathology, has its antecedents in laboratory and clinical studies of hemoglobin. Over the last century, knowledge of the genetics, functions, and diseases of the hemoglobin proteins has been refined to the molecular level by analyses of their crystallographic structures and by cloning and sequencing of their genes and surrounding DNA. In the last few decades, research has opened up new paradigms for hemoglobin related to processes such as its role in the transport of nitric oxide and the complex developmental control of the alpha-like and beta-like globin gene clusters. It is noteworthy that this recent work has had implications for understanding and treating the prevalent diseases of hemoglobin, especially the use of hydroxyurea to elevate fetal hemoglobin in sickle cell disease. It is likely that current research will also have significant clinical implications, as well as lessons for other aspects of molecular medicine, the origin of which can be largely traced to this research tradition.

Simulating hemoglobin history.

In one of the truly classic works in anthropological genetics, Frank Livingstone established the interrelationships between agriculture, mosquito ecology, malaria, and, consequently, the frequencies of sickle cell hemoglobin in West Africa. A major inference from Livingstone's study was the recency of malaria as a selective agent in human populations, only becoming significant after the adoption of agriculture in the last few thousand years. Clines of the abnormal hemoglobin alleles might therefore represent continuing waves of advance of adaptive alleles. In order to model the complex interaction of several hemoglobin alleles, selection, and gene flow spreading adaptive mutants, Livingstone turned to computer simulation. Numerous insights concerning the competitive increase of different alleles (hemoglobins S, C, and E and thalassemia), the rate of allele spread under different migration scenarios, including the potential importance of long-range migration, came out of these studies. These experiments also stimulated others to search for mechanisms that might increase the diffusion rate of hemoglobin variants, including kin-structured migration and epidemic disease selection. Recent molecular studies have substantiated major aspects of Livingstone's work (including the recent origin of falciparum malaria) and posed challenges to some of his assumptions (such as the number of mutations to hemoglobins S and E). But whatever the fate of his specific hypotheses, his emphasis on the interaction of genetics, ecology, and culture stands as a model for the anthropological approach to the understanding of human variation and evolution.

Historical review: the carbon monoxide diffusing capacity (DLCO) and its membrane (DM) and red cell (Theta.Vc) components.

The single breath carbon monoxide diffusing capacity (DLCO sb), also called the transfer factor (TLCO), was introduced by Marie and August Krogh in two papers (Krogh and Krogh, Skand. Arch. Physiol. 23, 236-247, 1909; Krogh, J. Physiol., Lond. 49, 271-296, 1915). Physiologically, their measurements showed that sufficient oxygen (by extrapolation from CO) diffused passively from gas to blood without the need to postulate oxygen secretion, a popular theory at the time. Their DLCO sb technique was neglected until the advent of the infra-red CO meter in the 1950s. Ogilvie et al., J. Clin. Invest. 36, 1-17, 1957 published a standardized technique for a 'modified Krogh' single breath DLCO, which eventually became the method of choice in pulmonary function laboratories. The Roughton-Forster equation (J. Appl. Physiol. 1957, 11, 290-302) was an important step conceptually; it partitioned alveolar-capillary diffusion of oxygen (O2) and carbon monoxide (CO) into a membrane component (DM) and a red cell component (theta.Vc) where theta is the DLCO (or DL(O2)) per ml of blood (measured in vitro), and Vc is the pulmonary capillary volume. This equation was based on the kinetics of O2 and CO with haemoglobin (Hb) in solution and with whole blood Hartridge and Roughton, Nature, 1923, 111, 325-326; Proc. R. Soc. Lond. Ser. A, 1923, 104, 376-394; (Proc. R. Soc. Lond. Ser. B, 1923, 94, 336-367; Proc. R. Soc. Lond. Ser. A 1923, 104, 395-430; J. Physiol., Lond. 1927, 62, 232-242; Roughton, Proc. R. Soc. Lond. Ser. B 1932, 111, 1-36) and on the relationship between alveolar P(O2) and 1/DLCO. Subsequently, the relationship between DL(O2) (Lilienthal et al., Am. J. Physiol. 147, 199-216, 1946) and DL(CO) was defined. More recently, the measurement of the nitric oxide diffusing capacity (DLNO) has been introduced. For DL(O2) and DLNO the membrane component (as 1/DM) is an important part of the overall diffusion (transfer) resistance. For the DLCO, 1/theta.Vc probably plays the greater role as the rate limiting step. A crucial question, the effect of unstirred plasma layers on the 'true' value of thetaCO in vivo, has not been resolved, but this does not detract from the clinical role of the DLCO sb (TLCO) as an essential test of lung function.

Hemoglobin-oxygen equilibria: retrospective and phenomenological perspective.

The origin of the concept of a molecular complex between oxygen and hemoglobin can be traced to Stokes, a century and a half ago. Subsequently, physicochemical concepts of equilibria provided a path to quantitative formulations of these ligand-receptor interactions. Then, it took a quarter of a century before a proper format was prepared in terms of four stoichiometric equilibria and their associated binding constants. Since then, experimental measurements of these stoichiometric binding constants have consistently disclosed that successive values of K(1) to K(4) are accentuated above those expected if every subunit of hemoglobin maintained the same, intrinsic, unchanging affinity for oxygen. An alternative analysis of the observed cooperative interactions has been obtained by extracting the roots of the polynomial of the stoichiometric binding equation and deriving an alternative binding equation containing constants that for O(2)-Hb are complex numbers. These constants have the dimensions and properties of equilibrium constants. They provide some novel phenomenological insights into ligand-receptor equilibria.