Sickle-Cell Anemia Hemoglobin: The Molecular Biology of the First “Molecular Disease”—The Crucial Importance of Serendipity

IN the spring of 1952 I sent 32 letters to England, trying to find a job in academic research. I was finishing my second postdoctoral year in the United States, working in Joseph Fruton's Biochemistry Department at Yale University. My topic was peptide chemistry, in particular the development of a novel end-group method by reductive methylation of the N-terminal α-amino group, followed by paper chromatography of the hydrolysate to detect a “basic” derivative of the end amino acid. The method worked and was published (Ingram 1950). To my knowledge, my method was never used by anybody, but of course I did not know this at the time! I was an organic chemistry Ph.D. with an interest in animal physiology developed through an excellent undergraduate course by Alistair Graham at Birkbeck College, London University, during the war years. My entrance to modern biology was, and still is, through the chemistry of proteins and peptides. Before Yale, I had spent a year with Moses Kunitz learning how to purify a yeast enzyme.1 Then I had that year at Yale with peptides. Now I was ready for new things. Kunitz and Northrop were among the first to purify and crystallize protein enzymes. I had become quite discouraged by my lack of success in finding a job back in England. After all, 3000 miles was and still is a long distance when you are job hunting. Quite by chance a new postdoc appeared just then in the Fruton lab. He was Herbert “Freddie” Gutfreund, straight from Cambridge, England. He knew well the Medical Research Council (MRC) Unit2 at the Cavendish Laboratory, where Max Perutz was director, and told me that Perutz, a protein X-ray crystallographer of note, needed a protein biochemist to place a “heavy atom” in a specific location in his hemoglobin (Hb) crystals. This approach would enable him to determine the phases of most of the X-ray reflections, the big problem in X-ray crystallography at that time. Freddie said “do apply” and I did, not expecting much. To my surprise, I was accepted for September 1952 and I was happily on my way back to England. Officially, the Medical Research Council Unit for the Determination of the Structure of Biological Molecules—rather a mouthful. The laboratories of the MRC Unit were modest, but modern. We had one large biochemistry room for about four people. Next door was a large office for Francis Crick and, later, for Jim Watson and Sydney Brenner. The suite also contained a modest office for Max Perutz and John Kendrew, both X-ray crystallographers working on the structure of hemoglobin and myoglobin, respectively. There also was a well-equipped machine shop where Tony Broad developed the rotating anode X-ray source, one basis for the supremacy of the laboratory; the much more powerful X-ray beam produced greatly reduced needed exposures of the vulnerable protein crystals. The X-ray cameras were in the basement, but the films on which the X-ray reflections were recorded had to be developed on the top floor (no elevators). My first impression of Francis Crick was of a research student rapidly oscillating between the basement and the top floor! The laboratory was, however, a close and congenial environment [Sidney Altman (2003) has written a Perspectives of the latter-day MRC-LMB]. The success of Kendrew and Perutz's X-ray studies soon brought a number of visitors, some, like Brenner, very long lived. We became overcrowded, always a good sign of a productive lab. Within a few years (in 1956?) we moved into a refurbished bicycle shed in the courtyard of the Cavendish laboratory, leaving the X-ray machines where they were. The new environment, although drafty, provided much-needed expansion space for protein chemistry, microbiology, and the large group of people reading X-ray films manually. Visitors from spacious labs in the United States found this a quaint place. However, it was in the bicycle shed that I first produced the early “fingerprints” of sickle-cell hemoglobin peptides, with the help of Leslie Barnett and Rita Fishpool, the technicians. I had long finished the project I was originally given by Max Perutz—to insert a single heavy atom into a unique position in the Hb molecule and then crystallize that derivative. This turned out to be straightforward, since the asymmetric Hb half-molecule, αβ, has only a single reactive sulfhydryl-cysteine side chain. This was readily reacted with p-chloromercuribenzoate. This heavy atom Hb derivative allowed Perutz and his student David Green to proceed to produce the first three-dimensional projection of a protein molecule (Green et al. 1954). This left me at a loose end. I became interested in the connection, if any, between the heme group of hemoglobin and any amino acid side chains. It was considered possible that the heme group might be covalently attached. Accordingly, I started preparing and characterizing tryptic hemoglobin peptides that contained the highly visible heme group. Nothing much came of this, because, as was realized later from the X-ray structure, there was no specific covalent link to a particular peptide. Instead, the precise location of the heme group in hemoglobin and myoglobin depended on noncovalent interactions with several specific amino acid side chains. Serendipity appeared again! Just at that time a very interesting visitor appeared at the MRC Unit: Tony Allison, bringing with him samples of sickle-cell anemia hemoglobin. Following Jim Neel's demonstration that sickle-cell anemia is caused by homozygosity for a sickle-cell gene, Tony had elegantly shown that the frequency for this gene, lethal when homozygous in an African population, was so surprisingly high because heterozygosity conferred protection against the endemic malarial parasite Plasmodium falciparum. The exciting story of this important discovery is described by Tony Allison in the previous issue of Genetics (Allison 2004). There were two reasons for Tony's coming to the MRC Unit: Max Perutz was interested in sickle-cell anemia hemoglobin, and he had the best X-ray equipment in the field. Perutz et al. (1951) had shown earlier that deoxygenated sickle-cell anemia Hb was much less soluble and formed “one-dimensional” crystals, leading to the well-known distortion of red cells in the disease; Tony wanted to put such “crystals/aggregates” of deoxygenated sickle-cell Hb into an X-ray beam and study the 3-D structure of these formations. As Allison (2004) describes, Linus Pauling had called the anemia a molecular disease and had shown with his colleagues that the Hb protein carried a chemical change, which was manifested as a charge difference for the whole protein, seen by Tiselius electrophoresis (Pauling et al. 1949). But was it one amino acid that was different, or two or three, or a whole group? Ordinary amino acid analysis at that time was too imprecise to decide. After all, it was only a very few years earlier that Sanger and Tuppy (1951) had convinced the world that a protein (insulin) was composed of a chain of amino acids, covalently linked, with a unique and defined amino acid sequence (see Stretton 2002). Things were moving fast in protein chemistry in those exciting days! Since I was already developing methods for characterizing large peptide fragments of proteins—and of hemoglobin and myoglobin in particular—Perutz and Crick suggested that I use these methods on sickle-cell anemia Hb and compare it with normal human hemoglobin (mine). I was able to use the remaining samples of sickle-cell anemia Hb left behind by Tony Allison, who had by then moved on. He had tried to take X-ray pictures of deoxygenated sickle-cell hemoglobin, a very difficult technical problem, because the hemoglobin to be mounted in capillaries had to be kept reduced; otherwise the “crystals” would redissolve. I had available the crucial abnormal protein on which to use the new concepts and techniques of Sanger (Sanger and Tuppy 1951; Sanger and Thompson 1952), which I was adapting to the much larger peptides I was preparing to examine. Hemoglobin, even the αβ half molecule, is 10-fold larger than either peptide chain of insulin. Sanger cleverly fitted together the amino acid sequences of a large number of very short overlapping peptides. To do the same with the much larger hemoglobin would have been a Herculean task. I needed to characterize larger peptide fragments, such as might be obtained by proteolytic digestion with trypsin, which gave some 26 peptides. In addition, I decided in the first instance not to attempt a full amino acid sequence of the whole protein, but to look first at the chemical behavior of these tryptic peptides and to sequence only those that became interesting in the sense of showing a difference between wild type and sickle-cell hemoglobin. In the event, that strategy proved to be enough to pinpoint a difference in chemical behavior and therefore in chemical structure. Sanger, dealing with very short peptides, was able to separate them cleanly by paper chromatography, then the most up-to-date method, using various solvents. My goal was twofold: first, to find a peptide fragment that showed an electrophoretic difference, as had the whole protein, and second, to show that the rest of the protein was likely to be the same, at least by the methods used. As so often experienced in molecular biology, we were doing chemistry! These considerations lay behind my evolving the method of “fingerprinting,” i.e., characterizing each peptide by its position on a two-dimensional map, a sheet of “blotting paper” (retold in Ingram 1989). I would digest with trypsin the two samples of protein, wild type and sickle-cell mutant, and then spot the resulting mixture onto a sheet of this paper moistened with buffer at pH 6.4 (near the isoelectric point for the whole protein). In stage 1, water-cooled electrophoresis under glass plates distributed groups of peptides with similar charge densities along a straight line. Stage 2 was partition chromatography at right angles, originally in a butanol:acetic acid mixture, which would resolve differences involving uncharged amino acid side chains. The resulting map or fingerprint of colorless peptides was “developed” by spraying with ninhydrin reagent to develop the purple color due to reaction with the α-amino group of each peptide (and also our fingers!). Figure 1 shows this result with the improved technique developed later by Corrado Baglioni (1961), a postdoctoral fellow. In these early days we argued that the differences seen for only one peptide were the only ones in the protein and that the rest of the sequence was normal. Actually, this conclusion was only as good as our techniques, which were still quite primitive. It remained for full amino acid sequencing of the HbS and wild-type HbA peptide chains by others to prove that our initial assumptions were correct. We were fortunate in that respect. With this caveat, we decided to go on and examine as many examples of other (inherited) abnormal hemoglobinopathies as we could. It happened that Herbert Lehmann of St. Bartholomew's Hospital in London, a doctor and good friend of Max Perutz's, was very interested in the epidemiology of sickle-cell anemia and related diseases (Perutz and Lehmann 1968). He soon moved to Cambridge. His freezer was stocked with an extensive collection of abnormal hemoglobin samples collected by him on his travels or supplied by his network of friends and colleagues. Without his support, much of our early and later work could not have been done. He became a constant source of inspiration and constructive criticism. —Fingerprints of hemoglobins A and S (improved method); photograph of ninhydrin-positive peptide spots on filter paper (Baglioni 1961). A very important phase of this work was the ability to replicate these fingerprints of HbS with five different samples of unrelated sickle-cell anemia patients before we could publish our work. Hermann Lehmann supplied these samples. Very soon I was able to hire Leslie Barnett and Rita Fishpool to help with the increasing demands for more and more fingerprints of new and exciting abnormal human hemoglobins. The isolation of interesting tryptic peptides from these hemoglobins led to amino acid analyses and Edman stepwise degradation to figure out the amino acid sequence and thereby the missense mutations and amino acid substitutions of the early abnormal hemoglobins. Two early graduate students joined me at the MRC Unit, John Hunt, now a professor in Hawaii, and Tony Stretton, now a professor at Madison. There was a “temporary interruption” when I went for a sabbatical leave to the Biology Department at the Massachusetts Institute of Technology (MIT) in the “other” Cambridge, where I have stayed for 45 years! Other graduate students and postdocs joined me there. The early work in England with sickle-cell anemia and with hemoglobin C and E diseases was later followed at MIT with more and more complicated inherited hemoglobinopathies and thalassemias. A whole new world of fascinating research had opened up! The driving force was the realization that we were chemically exploring the mechanisms of Mendelian inheritance and the evolution of gene clusters. The important conclusion from our earliest experiments was that a simple Mendelian trait resulted in the substitution of just one amino acid. This realization occurred just at the time when Crick and Brenner were figuring out the fundamental properties of the genetic code for an amino acid sequence. Our finding of a single amino acid substitution made impossible some coding schemes, then popular in the days before DNA sequencing, that involved an overlapping triplet code. Such a code was made impossible because in that scheme a single base substitution in DNA would affect not one but several adjacent amino acids, depending upon the particular coding number and pattern of overlap. —Amino acid sequences of the N-terminal peptides of the β-chain of hemoglobins A, S, C, and GSan José; the last should not be confused with hemoglobin GPhiladelphia, which is an α-chain mutation. Quite quickly the amino acid substitutions were seen to demonstrate the molecular basis at the phenotypic level of allelism, heterozygosity, and homozygosity. They illustrated codominance, evolutionary changes after gene duplication, and unequal crossing over. Through fingerprinting analysis of amino acid sequences of hemoglobins, Corrado Baglioni showed unequal crossing over in the abnormal HbLepore, which contains a β-like peptide chain that was β-chain and (Baglioni different of mutations were found in the hemoglobins by other mutations and The were important in thalassemias. The which were in were often found to They also were more than to to The which are α-chain are very and also the molecular basis of a of hemoglobinopathies could be by amino acid sequences of hemoglobins, originally with the fingerprinting techniques, but soon through the use of more methods of peptide diseases than sickle-cell the and hemoglobinopathies single amino acid missense They also are much more than but the of which is less well the other end of the the abnormal hemoglobins and have an amino acid this a of two but missense or is it due to a a long time As as sickle-cell anemia is it was at one time that an high of might the high HbS study showed 2 among some with sickle-cell anemia were not sickle-cell anemia i.e., were not for the mutation. This would an high It later turned out that between were quite in that particular and were not the of the one a of for this In this a single is In last Tony Allison (2004) the fascinating story of how he the of in the that the high frequency for the so lethal when at least in where it is by years brought many from the malarial of to the of and to the of in the of was with the of has always been of In many later, that the frequency of the HbS was only on but on the In the frequency of the was and that the two came from the same The study shows that greatly the frequency of sickle-cell it to have on the frequency of the there is some other for the high frequency of the in of the in human hemoglobin (Ingram as in We also needed to know for which of the two different peptide chains in hemoglobin, the or the was involved in the sickle-cell mutation. both were since at that time the amino acid sequences of the two chains were not to our it turned out that only the β-chain carried the sickle-cell mutation. We could now with the and of to peptide Our finding of a single one amino acid greatly the of a between the genetic molecule structure had just been and the protein peptide chain by Sanger for A good now also had to be a good now to the peptide chains of the human the and the and the and This was to missense mutations to particular chain The work was by at and by and at the it was by many The of these peptide chains was in the structure of the human (and gene the α-chain gene the and and the β-chain gene the and These gene are still used to study gene and that during of the hemoglobin chains. The point in time of a gene is by a (Ingram Our analysis of the amino acid sequences of and human hemoglobin peptide chains led to an evolutionary scheme for the that the peptide chains. to this was the of the close sequence and between the myoglobin and the hemoglobins. We used a of sequence evolution in which we put to each other in evolution those peptide chains with the amino acid We that evolution occurred gene duplication, not a new followed by evolution of the resulting This evolution was not quite however, since and β-like could only the of the that they able to This was to the between that the very The scheme was well and to many other evolutionary The sickle-cell disease in and but not in the since it only which are not in or red The disease after years of A in the on the other would affect hemoglobins in the and as well as in the of human since the α-chain is to the human hemoglobins at Tony Stretton and I to some to the that among the of the various of This is a group of inherited hemoglobinopathies in which there are only normal hemoglobin peptide but a in hemoglobins more in one or hemoglobin peptide chain. 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