Background for two articles by Vernon M. Ingram (1958, 1959)
Abnormal Human Hæmoglobins:
I. The Comparison of Normal Human and Sickle-Cell Hæmoglobin by "Finger Printing."
Biochem. Biophys Acta 28, 539 - 545 and
III. The Chemical Difference between Normal and Sickle-Cell Hæmoglobin.
Biochem. Biophys. Acta 36, 402 - 411
Pauling and coworkers (1) demonstrated that hemoglobin in the red blood cells of patients with sickle-cell anemia differed from normal human hemoglobin by having 2 - 4 more net positive charges. That work represented a significant advance. Not only did it convincingly show that a specific gene could influence the chemical structure of a specific protein, it suggested that the altered chemistry, in a sense, caused sickle-cell anemia. Identifying the small number of specific chemical difference in hemoglobin, a huge molecule, immediately became and obvious challenge, without an obvious solution. Such challenges attract outstanding scientists in creative supportive environments.
In 1956, Vernon Ingram was a young biochemist working in Cambridge England. He had the laboratory next door to where Watson and Crick had recently solved the structure of DNA (2). His boss and director of the Medical Research Council (MRC) Laboratory of Molecular Biology, Max Perutz, had solved the three-dimensional structure of hemoglobin by X-ray crystallography (3) and was a Nobel Laureate (4). Ingram had access to samples of sickle-cell hemoglobin brought to Perutz by A. C. Allison who had recently shown that the sickle-cell gene confers resistance to malaria (5). Ingram was in the right place at the right time and was up to the challenge of chemically dissecting hemoglobin.
Ingram's approach was to divide the problem into smaller parts. Using the enzyme trypsin, he hydrolyzed specific peptide bonds, separated and compared the chromatographic and electrophoretic properties of the resulting peptide fragments, and focused his attention on the chemical structure of the single peptide from HbA and HbS that differed. Thus the first part of the hemoglobin structure (other than the heme) to be determined resulted from an interest in sickle-cell anemia. If you would like to have a computer generate the peptides from a hemoglobin amino acid sequence, copy the sequence, e. g. human hemoglobin alpha chain or beta chain, from the National Center for Biotechnology Information, and paste it into the Cutter program.
Ingram originally published his results in two short papers in Nature (6,7). Those articles contain little experimental detail and actually misinterpret some of the experimental evidence. Fortunately the essential facts were correct and Ingram had the chance to correct the errors of interpretation in the articles you will read.
The study of hemoglobin touches on many of the dimensions of biochemistry. That is why hemoglobin provides a convenient theme for an introduction to biochemistry course. Ingram's articles introduce several new dimensions to this course. Organic chemistry developed by others was used to elucidate the structure of peptides. Techniques such as dialysis, paper chromatography, paper electrophoresis, and preparative centrifugation have general utility in biochemical work. I hope that the context makes these methods more relevant and memorable than when presented in isolation. Dr. Ingram visited the C-342 class on May 5, 1995.
1.* Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C. (1949) Sickle Cell anemia, A molecular disease. Science 110, 543 - 548.
2. Ingram, V. M. (1989) A case of sickle-cell anæmia: a commentary by Vernon M. Ingram. Biochem. Biophys. Acta 1000, 147 - 150. (This is an autobiographical article commemorating significant articles published in BBA on the occasion of reaching volume 1000)
3.* Perutz, M. F., Muirhead, H., Cox, J. M., and Goaman, L. C. G. (1968) Three-dimensional Fourier synthesis of horse oxyhæmoglobin at 2.8Å resolution: the atomic model. Nature 219, 133 -139.
4. Perutz, M. F. (1963) X-ray analysis of Hemoglobin. Science 140, 863 - 869. (This is a copy of Perutz's Nobel address delivered on 11 December 1962)
5.* Allison, A. C. (1954) Protection afforded by sickle cell trait against subtertian malarial infection. Brit. Med. J. 1, 290 - 294.
6. Ingram, V. M. (1956) A specific chemical difference between globins of normal and sickle-cell anæmia hæmoglobins. Nature 178, 792 - 794.
7. Ingram, V. M. (1957) Gene mutations in human hæmoglobin: the chemical difference between normal and sickle cell hæmoglobin. Nature 180, 326 - 328.
* Articles that can be found in the course reader.