Hemoglobin research and the origins of molecular medicine

Abstract

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.

Figures

Alan N. Schechter

Figure 1

The X-ray determined structure of the hemoglobin molecule and a representation of its very high concentration in the erythrocyte. (A) The arrangement of the α-helices (shown as tubes) in each αβ unit—one on the left and one, 180° rotated, on the right—is shown, as are the 4 heme groups with their iron atoms where gas molecules bind. The site of the sickle mutations on mutant β-chains as well as the β93 conserved cysteine residues is also shown. Hemoglobin molecules in the red blood cell, shown in an inset on the right, are very tightly packed (at a concentration of approximately 34 g/dL) and have little access to solvent; this allows efficient oxygen transport by each cell but also affects the chemical behavior of the molecules, such as promoting sickle cell hemoglobin polymerization upon slight deoxygenation. (B) A representation of the quaternary structural changes in the hemoglobin tetramer, in a top-down view, in the transition from the oxy conformation (left) to the deoxy conformation (right). The iron atoms shift relative to the planes of the heme groups and a central cavity between the β-chains opens, facilitating 2,3 BPG binding. These diagrams are based on drawings of Irving M. Geis. Illustration by Alice Y. Chen.

Figure 2

A diagram of the proposed evolutionary relationships of the human globin proteins as inferred from sequence analyses. NGB, neuroglobin; CYGB, cytoglobin; MB, myoglobin. Reprinted from Pesce et al (EMBO Rep. 2002;3:1146-1151) with permission. Illustration by Alice Y. Chen.

Figure 3

A photograph of Max F. Perutz (1914-2002) demonstrating an early model of the structure of hemoglobin. He devoted more than a half-century to the study of the detailed molecular structure of hemoglobin but was always directly concerned with the relevance of his work to understanding its function and its role in human disease. Courtesy of the Medical Research Council (London, United Kingdom).

Figure 4

A representation of nitric oxide (NO)/hemoglobin reactions in the arterial microcirculation. Reactions that appear to predominate under physiologic conditions (center), as well as pathologic lesions due to hemolysis (right) and results of high or pharmacologic levels of NO (left) are indicated. Under basal conditions, NO (a short-lived free radical) produced by endothelial NO synthase enzymes largely diffuses into surrounding smooth muscle to activate soluble guanylyl cyclase (sGC) to produce cyclic GMP and regulate vascular tone. The interactions of NO with red cells under these conditions seem to be limited by several barriers to diffusion, at the red cell membrane and streaming of plasma near the endothelium. With hemolysis (or with administration of hemoglobin-based blood substitutes), cell-free oxyhemoglobin acts as an efficient scavenger of NO, causing vasoconstriction and perhaps pathological organ conditions. When endogenous NO levels become very high, or when it is administered by inhalation or by infusion of nitrite ions or other NO donors, reactions in the plasma and within erythrocytes become very important. Reactions with oxygen will tend to oxidize NO to nitrite and nitrate. Reactions with plasma molecules will form thiol (SNO) compounds and other species; plasma nitrite can also be reduced by endothelial xanthine oxidoreductase (XOR) to NO. Small amounts of SNO-Hb form, but its function is not at all clear. Nitrite from the plasma may enter the red cell or be formed in the cell itself, where reactions with hemoglobin and the ascorbate cycle can reduce it to NO. Although the prevalent reactions with oxyhemoglobin to form methemoglobin and nitrate tend to destroy NO bioactivity, these other reactions may allow its preservation and modulation for physiologic functions. Adapted from Schechter and Gladwin (N Engl J Med. 2003;348:1483-1485), with permission. Illustration by Alice Y. Chen.

Figure 5

The genomic structure of the clusters of α-like and β-like globin genes, on chromosomes 16 and 11, in human beings. The functional α-like genes are shown in dark blue and the pseudogenes are in light blue; 2 of these (μ and θ-1) code for small amounts of RNA. The functional β-like genes are shown in light green. The important control elements, HS-40 and the LCR, discussed in the text, are also shown at their approximate locations. The α-gene cluster is approximately two thirds of the length of the β-gene cluster; it is transcribed from telomere toward centromere, the opposite of the β cluster. The various hemoglobin species that are formed from these genes, with their prime developmental stages, are shown in the lower part of the figure. Illustration by Alice Y. Chen.

Figure 6

The timeline of the expression of the human globin genes from early stages of fetal development to the changes that occur at birth and in the first year of life. Also shown are the major sites of erythropoiesis and the types of hemoglobin-containing cells during these periods. These analyses are largely based on observations of clinical samples made by Huehns et al in the 1960s; the figure is reprinted from Wood (Br Med Bull. 1976;32:282) with permission. Illustration by Alice Y. Chen.

Figure 7

A diagram of the postulated effect of hydroxyurea in inhibiting hemoglobin S-polymerization, by increasing hemoglobin F levels (shown as 25%) in each sickle erythrocyte and thus decreasing the degree of microvascular obstruction at any oxygen level. The sparing effect of hemoglobin F, greater than that of hemoglobin A, occurs because the mixed hybrid α2βSγ that forms inside the red cell does not enter the polymer phase. Adapted from Schechter and Rodgers (N Engl J Med. 1995; 334:333-335), with permission. Illustration by Alice Y. Chen.

Figure 8

A simplified model of the developmental control of β-globin gene transcription at the embryonic, fetal, and adult developmental stages (see Figures 5 and 6). The sequential interaction of the LCR and trans-acting factors, such as GATA and EKLF, with the promoters of the several globin genes are shown as are putative cis-acting silencers that may down-regulate expression for “switching.” Evidence for silencers is stronger for the ε-globin gene, but it has been assumed that they would also affect the γ-globin genes. Levels of expression are indicated by the lengths of the arrows above each gene. In 3 dimensions, these interactions would be represented by loops of DNA with bridging transcription and chromatin proteins between the LCR and the genes, now called active chromatin hubs. A more detailed model would include many other transcription factors, modifications of chromatin structure, and the possible role of microRNA molecules as well as many post-transcription control mechanisms. Illustration by Alice Y. Chen.