Biochemistry - Chemical Reactions in Living Cells, Volume 1 - D. Metzler 1980

How molecules join together
Cooperative conformational changes
Comparative biochemistry of hemoglobin: abnormal hemoglobins

Even humans have several types of Hemoglobin. In addition to Myoglobin and adult hemoglobin A (a₂β₂), the minor hemoglobin A₂ (a₂δ₂) is also known. Human fetal Blood contains another type of hemoglobin—hemoglobin F (a₂γ₂). In the presence of 2,3-diphosphoglycerate, it exhibits a higher affinity for oxygen than hemoglobin A, which facilitates its primary function of supplying the fetus with oxygen. Several months after birth, hemoglobin F disappears and is replaced by hemoglobin A. Human Hemoglobins differ in their Amino Acid Composition and sequence. In other species, The amino acid composition of hemoglobins varies even more significantly. Interactions between subunits also vary, and one type of hemoglobin, erythrocruorin found in certain invertebrates, has 192 subunits [79].

What do all hemoglobins have in common? First of all, they share the same folding pattern of polypeptide chains around identical (or very similar) heme groups. However, the most striking fact is that, despite the pronounced uniformity of the overall Structure of all hemoglobins, there are only nine invariant amino acid residues and one nearly invariant residue. These ten residues are enclosed in rectangular boxes in Fig. 4-17. Two glycines (or alanines) at positions B-6 and E-8 are invariant because close contact between helices B and E does not allow larger amino acid residues to occupy these positions. Proline C-2 provides the bend in the molecule. Four other invariant residues are directly associated with the heme group. Two of them, His E-7 and His F-8, are "heme-linked" histidines. The ninth residue, Tyr HC-2, already discussed in Section 5.a, plays a major role in Cooperative oxygen binding. Only Lys H-9 is located on the outer surface of the molecule. The reasons why this residue is invariant remain unclear [80].

Box 4-G

Sickle-Cell Anemia

A Gene, relatively common in individuals of African descent, causes (when homozygous) a severe and often fatal disease known as sickle-cell anemia³. In 1949, Pauling, Itano, and coworkers discovered that the hemoglobin of patients with sickle-cell anemia has unusually high electrophoretic mobility^b. Later, in 1957, Ingram^v developed the peptide mapping method (Chapter 2, Section 3.2, Fig. 4-20) and applied it to study hemoglobin. He cleaved the hemoglobin molecule with Trypsin into 15 Peptides and separated the resulting mixture using Electrophoresis and Chromatography. He was able to demonstrate that the abnormality characteristic of sickle-cell hemoglobin (hemoglobin S) is localized in the ß-chain (at the sixth position) (Fig. 4-17). Glutamic acid, which occupies this position in normal hemoglobin, is replaced by valine in hemoglobin S. This was the first instance where the cause of a genetic disease was traced to THE MOLECULAR LEVEL, revealing a single amino acid substitution in a specific protein.

At low partial pressures of O₂, hemoglobin S crystallizes within erythrocytes. Crystallization disrupts The structure of THE RED BLOOD Cells, causing them to assume a sickle shape and break down easily, leading to anemia. The appearance of a hydrophobic amino acid residue, valine, at the 6th position located near the end of the molecule promotes The formation of a new binding site. As a result, hemoglobin tetramers associate to form long microtubular structures^g that crystallize inside the erythrocytes.

What is the reason for The high frequency of the sickle-cell gene (approximately 3 million Americans are known to be carriers)? The fact that this gene has "survived" and occurs most frequently among African populations can likely be explained by the idea that, alongside its harmful effects, it also conferred a certain protective advantage. Specifically, the malaria parasite, which has historically caused high human mortality, spends part of its life cycle within erythrocytes (Fig. 1-7). It turns out that in red Blood Cells containing both hemoglobin A and hemoglobin S, conditions for the growth of the malaria parasite are less favorable than in cells containing only hemoglobin A. Consequently, heterozygous carriers of the sickle-cell gene survived malaria epidemics, although this came at a high cost—one-fourth of their offspring perished from sickle-cell anemia.

What are the Prospects for treating this disease? Lifespans can be prolonged through blood transfusions, but these measures are not curative. Recent studies have shown that cyanate reacts with the terminal amino group of valine in the ß-subunits of hemoglobin S, which reduces the likelihood of sickling. The reaction is described by the following equation:

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The drug was tested on humans with great caution, but proved to be too toxic. Nevertheless, there is hope that new therapeutic agents can be found to inhibit the crystallization of hemoglobin S.

^а The much more numerous heterozygotes carry one hemoglobin S gene but have, at most, minor problems.

^б Pauling L., Itano H. A., Singer S. J., Wells I. C., Science, 110, 543—548 (1949).

^в Ingram V. M., Nature (London), 180, 326—328 (1957).

^г Wishner В. C., Ward К. B., Lattman E. E., Love W. E., JMB, 98, 179— 194 (1975).

^д Harkness D R., Trends Biochem. Sсi., 1, 73—76 (1976).

Many variations in human hemoglobin structure result from Mutations. It has been established that one in every 600 individuals carries a mutant hemoglobin in which typically one amino acid is replaced by another without any apparent consequences (Fig. 4-20). However, substitutions near the heme group often adversely affect oxygen binding, while substitutions in one of the subunit interfaces can impair cooperative interactions between them [81]. One of the most widespread abnormal hemoglobins with severely compromised function is hemoglobin S, found in individuals suffering from sickle-cell anemia (Box 4-G). In hemoglobin S, the glutamic acid residue at the 6th position of the ß-chain is replaced by a valine residue. Interestingly, the substitution of this same glutamic acid residue with a Lysine residue yields hemoglobin C, the presence of which is not associated with the severe pathological disorders seen in sickle-cell anemia. A number of other identified and characterized substitutions are shown in Fig. 4-17.

FIG. 4-20. Peptide maps of human hemoglobins. Denatured hemoglobin was digested with trypsin, and the resulting mixture of 28 peptides was separated on paper by electrophoresis (in the horizontal direction; anode on the left) and chromatography (in the vertical direction). Peptide locations were identified by spraying the paper with ninhydrin or Specific Reagents for Histidine or Tyrosine residues. A. Peptide map of hemoglobin A from a healthy adult. B. Peptide map of hemoglobin S. It is easy to see that one histidine-containing peptide (1) is missing, whereas a new peptide (2) appears. It contains the first 8 residues of the N-terminus of the protein subunit chain. (Lehman Н., Huntsman R., Man’s Haemoglobin, North-Holland, Amsterdam, 1974.)

Of particular interest is the family of M hemoglobins. The presence of such hemoglobin in the blood leads to severe disorders; only heterozygotes for this abnormal trait survive. Blood in these cases is dark because the iron in half of the hemoglobin M subunits is irreversibly oxidized to the ferric state (methemoglobin). In normal blood, the methemoglobin content does not exceed ~1%. Normally, methemoglobin is reduced by a specialized methemoglobin reductase system (Box 10-A), whereas M hemoglobins are not reduced. All five M hemoglobins possess substitutions at sites located near the heme group. In four of them, one of the heme-linked histidines (F-8 or E-7) in either the a- or ß-subunit is replaced by tyrosine. In the fifth, valine-67 in the ß-subunits is replaced by glutamic acid. Two M hemoglobins with substitutions in the a-subunits (M_Boston and M_Iwate) are "frozen" in the T (deoxy) conformation; they exhibit low oxygen binding affinity and bind oxygen noncooperatively.

In hemoglobin Rainier¹, the invariant tyrosine HC-2 is replaced by Cysteine. The oxygen affinity of this hemoglobin is extremely high, but cooperativity is absent. Hemoglobin Kansas, in which asparagine-102 in the ß-subunit is replaced by Threonine, also lacks cooperativity and has a very low oxygen affinity, whereas hemoglobin Richmond, in which the same asparagine is replaced by lysine, Functions normally. In hemoglobin Hiroshima, the C-terminal histidine in the ß-subunit is replaced by aspartic acid. This C-terminal histidine is one of the Amino Acids responsible for the Bohr proton release; consequently, in the mutant hemoglobin Hiroshima, oxygen affinity is increased threefold, and the Bohr effect is halved [82].

¹ The names of most abnormal hemoglobins are generally derived from the cities or hospitals where they were first discovered. Initially, researchers attempted to designate these hemoglobins with various letters, but when their number grew so large that the alphabet was exhausted, they reverted to traditional designations. The English names used in scientific literature have been retained in the text and in Fig. 4-17 so that the reader can precisely identify the specific hemoglobin being discussed — Transl. note.