Science in Christian Perspective

Hemoglobin Structure and the 
Biogenesis of Proteins. 
Part II. Significance of Protein Structure to the Biogenesis of Life
GORDON C. MILLS
Dept. of Human Biological Chemistry and Genetics
University of Texas Medical Branch Galveston, Texas

From: JASA 27 (June 1975): 79-82.

In Part I (Journal ASA, March 1975), the author has reviewed structure-function relationships for the hemoglobin molecule, with particular emphasis on the properties of mutant hemoglobins of man, In Part II, the relation of these studies to theories for the biogenesis of life is discussed. It is concluded that protein function is a consequence of a unique arrangement of the individual amino acids, and that this arrangement could not be achieved by chance. It is noted that data on protein stability is not in accord with views that proteins might survive for extremely long periods of time. Adverse effects of inactive protein molecules on cellular function are also noted. The data strongly favor the view that "design" or "intelligence" must be involved in the beginning of life.

The review of properties of abnormal hemoglobins suggests several aspects that are significant in relation to theories for the biogenesis of life. A tenet advanced by various workers, and especially by Fox and his coworkers', has been that a random combination of amino acids would produce a protein with minimal, but adequate function. Presumably, this protein, in the absence of degradative organisms, would remain functional until all other proteins, nucleic acids, etc., necessary for primitive life were also produced. Among the implications of this hypothesis are:

(1) The function of a protein as a catalyst, oxygen carrier, etc., is not uniquely dependent upon amino acid sequence. Also numerous bonds other than the peptide bond linking the amino group and the C₁-carboxyl group could he tolerated. For example, peptide bonds might utilize the amino group of lysine, the C₄-carboxyl group of aspartic acid or the C₅--carboxyl group of glutamic acid.
(2) In the absence of organisms or enzymes produced by organisms, a protein molecule would retain functional activity for long periods of time (thousands of years?).
(3) All of the functionally inactive protein molecules in the protein "soup" would not adversely affect the action of those proteins that did have functional activity.
(4) Minimal functional activity of the various essential proteins would be all that would he necessary to sustain life.
(5) Out of all the randomly produced protein molecules, one is selected and ultimately serves as the archetypal globin chain, the precursor of the various polypeptide subunits of globin for all living organisms which utilize a heme protein as an oxygen carrier.

Protein Function and Amino Acid Sequence

Let us consider the relation of protein function to amino acid sequence. Is it plausible to suggest that a random arrangement of amino acids would produce a protein that would function in the transport of oxygen as does hemoglobin? The studies summarized earlier indicate that the polypeptide chains making up the hemoglobin molecule are indeed unique, and that the properties of these polypeptides are a consequence of their amino acid sequence. A recent tabulation indicated that 41% (49 out of 112) of the known hemoglobin mutants have impaired functions². For mutants where the amino acid substitution involved an internal portion of the molecule or contacts between subunits, 89% (41 of 40) had impaired function. It seems likely that the means utilized for detection of mutant hemoglobins would introduce some bias toward detecting hemoglobins with abnormal function. Nevertheless, the conclusion is clear that a single amino acid change in the hemoglobin molecule will, in a high percentage of eases, result in diminished funetion^o.

A unique structure for hemoglobin is also indicated if one examines the known variations in amino acids at each of the positions of the beta, gamma and delta chains of the vertebrate hemoglobins³. There are 40 positions in the poly peptide chain where there may he 2 alternative amino acids, 23 positions with 3 alternatives, 13 positions with 4 alternatives, 11 positions with 5, 5 positions with 6, 3 positions with 7, and 1 position with 8. Amino acids in the remaining 50 positions are identical in the species studied. Theoretically, there could he 20 possible alternatives for each position in the molecule. As hemoglobins from additional species of vertebrates are studied, the number of alternatives for amino acids at each position of the polypeptide chain will undoubtedly be increased somewhat. Nevertheless, the trend of the data clearly supports the view that there can he only limited replacement of amino acids without loss of function.

The structure of the polypeptide chains of the hemoglobin molecule is a consequence of design and not simply of chance combinations of amino acids. Murray Eden and George Wald also conclude that chance combinations of chance mutations cannot explain the unique structure of the hemoglobin molecule.

It is also important to note that every peptide linkage in the globin poly peptide chain involves the a-amino group and the C₁-carboxyl group. The e-amino group of lysine, the C₄-carboxyl of aspartic acid, and the Q₅-carboxyl of glutamic acid are not involved in peptide linkages even though these amino acids occur frequently in the molecule (for the human beta chain: aspartic acid, 7 times; glutamic acid, 8 times; and lysine, 11 times). Also, every optically active amino acid in the globin polypeptide chain is of the L- configuration. This implies optical purity of the starting materials and no raeemization during the formation of the amino acid polymer. These characteristics of the hemoglobin molecule are not found in the proteinoid produced from amino acids by thermal polymerization¹.

The studies cited are in accord with the view that the structure of the polypeptide chains of the hemoglobin molecule is a consequence of design and not simply of chance combinations of amino acids. Any plausible theory for the biogenesis of proteins must provide an explanation for the ordered sequence of amino acids in the protein molecule. Murray Eden and George Wald have briefly considered the relation of hemoglobin structure to evolutionary theory⁴. They also conclude that chance combinations or chance mutations cannot explain the unique structure of the hemoglobin molecule.

Protein Stability

A second aspect of the properties of hemoglobin is that related to protein stability. As noted above, proponents of the random combination theory for the formation of proteins presume that a protein molecule will retain functional ability for long periods of time. When compared to many enzymes, hemoglobin would he considered a relatively stable protein. Nevertheless, hemoglobin A in solution gradually is oxidized to methemoglobin. If the heme is removed, the globiu becomes increasingly unstable and precipitates. Within the red cell there are protective enzyme systems that maintain hemoglobin in the functional state. Methemoglobin reductase is continually involved in reducing methemoglobin (ferric iron) to ferrohemoglobin⁵. Enzymes linked to glutathione peroxidase are essential for the protection of hemoglobin from oxidative breakdown°. In the intact metabolically active red cell, normal hemoglobin retains its functional ability for the life span of the cell (ca. 120 days). However, when the hemoglobin is abnormal, or when there is an abnormality in one of the enzymes involved in the protection of hemoglobin, a pathologic condition often results. The unstable hemoglobins illustrate this point very well. The amino acid modification causes instability in the molecule, the globin precipitates within the red cell, and the precipitate often attaches to the membrane⁷. With some mutants, the consequences of this globin precipitation within the cell are very severe (e.g., Hb Sabine)^8,9. There are also a wide variety of drug-induced hemolytic anemias¹⁰, where a defect in one enzyme in the protective enzyme sequence permits hemoglobin damage and precipitation within the cell. In another type of hemolytic anemia, the formation of either beta or alpha chains within the maturing erythrocyte is impaired². Under these circumstances, there is a formation of a hemoglobin from four like polypeptide chains; i.e., Hb a₄, HI) H (b₄) or Mb Barts (g₄). These hemoglobins are functionally abnormal and are unstable. Hb H and Hb a are quite deleterious to the cell due to intracellular precipitation and fib Barts appears to be lethal to the fetus. This indicates that not only must a tetrameric structure be formed, but that we must have the correct subunits in the tetramer.

The studies summarized above clearly show that even normal hemoglobin is only moderately stable. In the absence of bacteria or enzymes, it would still deteriorate within several months. If the hemoglobin is modified by substitution of certain amino acids (Part I, Table 2), it becomes unstable. As noted above, the stability of hemoglobin does not appear unusual when compared with other proteins. Consequently, studies of hemoglobin stability are not consistent with hypotheses for biogenesis of protein which appear to require a protein to remain functionally active for very long periods of time, while all other essential components for life were being formed.

If one does permit the intervention of intelligence or design, one could make reasonable speculations, but with no apparent way of knowing whether the hypotheses are valid.

Effect of Inactive Protein

Another tenet of the theory for the biogenesis of proteins is that all of the totally inactive proteins (99.9% of the total ?) would have no adverse affect on those that do have biological activity. In the hemoglobin mutant, Hb Sabine, the abnormal protein in solution within the red cell constitutes 10 to 15% of the total hemoglobin. Possibly an equivalent amount is precipitated within the cell. No defective enzymes have been demonstrated in these cells and the amount of normal hemoglobin within the cells appears to be adequate for oxygen transport. Nevertheless, the life span of the red cells is markedly shortened and this appears to be due to precipitation of the unstable hemoglobin within the cell. These studies provide experimental evidence that a living cell cannot tolerate large amounts of nonfunctional intracellular protein, especially if the latter tends to precipitate and bind to membranes.

Minimal Protein Function and Life

The implication that minimal function of a protein would he all that would be necessary to sustain life is difficult to prove or to disprove. With enzymes, the enzymic activity in most eases is greater than is necessary for normal metabolism, and one could argue that diminished activity would not necessarily be deleterious to the cell. However, there are a wide variety of known genetic disorders due to enzyme deficiencies. In many of these cases, the mutant enzyme retains 5% or more of normal activity, yet this reduced activity is not sufficient to maintain cellular metabolism. If one enzyme with markedly reduced function can cause the severe consequences noted in these genetic disorders, it does not seem possible that a cell could survive with all proteins exhibiting only minimal function. Also, one of the primary characteristics of life is that enzymic pathways are under metabolic control. A portion of this control is exercised by enzymes that have a control site or sites on the enzyme molecule that are distinct from the catalytic site. Some type of control seems to be a prerequisite for life. The postulation of chance formation of protein molecules with enzymic function implies that there would he no control of enzymic pathways. This lack of control would he totally contrary to life as we see it exemplified in living cells today. Although some primitive enzymes might serve in metabolic pathways, in other cases, a more sophisticated enzyme (e.g., one with narrow substrate specificity, allosteric sites, etc.) would be required. One might make the comparison of the tetrameric hemoglobin molecule to a more sophisticated enzyme. The properties of hemoglobin are dependent not only upon the oxygen-binding site, but its physiologic function is dependent upon the proper interaction between the four subunits and also upon the binding of 2, 3-diphosphoglycerate. Although one might consider the monomeric myoglobin molecule as a more primitive oxygen carrier than hemoglobin, careful inspection of the data indicates a complex structure for myoglohin. Its properties are dependent upon a region of specific amino acids surrounding the heme, and also upon having appropriate intrachain contacts between R-groups of the component amino acids. Consequently, the hypothesis that only minimal protein function would be required for primitive life is not supported by the available experimental evidence.

Selection and Reproduction of an Archetypal Globin

The question of how one functionally active protein molecule could be selected from the tremendous numbers of non-functional molecules and be utilized for the reproduction of further like molecules is totally answered. Instead of presuming that proteins were the first molecules that were formed, it would appear more reasonable to propose that the first molecules formed were nucleic acids. The transfer of information for amino acid sequence could then proceed by basepairing of the purine and pyrimidine bases. The major problem, of course, is that polynucleotide synthesis requires a protein enzyme as a catalyst. Also, the synthesis of the mononucleotide budding blocks (nucleoside triphosphates) for polynucleotide synthesis requires enzyme catalysis. Consequently, if one chooses to make the presupposition that no "intelligence,, or "design" is involved in the biogenesis of life, the problems appear to be insurmountable. If one does permit the intervention of "intelligence" or "design", one could make reasonable speculations, but with no apparent way of knowing whether the hypotheses are valid.

One additional aspect of hemoglobin structure that merits attention is the lack of heterogeneity in the polypeptide chains that is found in a particular species. If the different hemoglobins are all derived from some archetypal precursor by mutations, deletions, chain fusion, etc., a marked heterogeneity in structure should be evident in every species. A small amount of heterogeneity has been noted in the glnbin molecule in recent studies¹¹. This would be consistent with some evolutionary modification of the pnlvpeptide chains with the passage of time. This should he distingnshed however, from the marked heterogeneity expected if all presently existing glohins were derived from a single archetypal precursor. The author has discussed this problem in evolutionary theory in more detail previously in relaton to the structure of cytochrome c¹², and the problem will simply be noted in this publication.

Footnote

^oThere is no indication that any of the mutant hemoglobin' have superior function when compared with HhA. Consequently, there would he no reason to suggest that natural selection would, at some time in the future, establish one of the mutant hemoglobins as the predominant type.

REFERENCES

Fox, S. W., Harada, K., Krampitz, G., and Mueller, C., Chem. Eng. News 48, No. 8,80 (1970).
2Stamatoyannoupnulos, C., Ann. Rev. Gen. 6, 47 (1972).
5Dayhoff, M. 0., Atlas of Protein Sequence and Structure 1972, National Biomedical Research Foundation, Silver Spring, Md., p. D-371/D-372.
⁴Eden, 81. in "Mathematical Challenges to the Neo-Darwinian Interpretation of Evolution", P. S. Moorhead and M. M. Kaplan, editors. Wistar Institute Press, 1967, p. 6-19.
⁵Jaffe, E. R. and Hsieh, H. S., Sem. Hemat. 8, 417 (1971). 
⁶Mills, C. C., Tex. Rep. Biol. Med. 21, 487 (1963).
⁷Jacob, H. S., Sem Hemat. 7, 341 (1970).
⁸Mills, C. C., Lcvin W. C. and Alpcrin, J. B., Blood 32, 15 (1968).
⁹MiIls, C. C., Alperin, J. B., Hill, F. L., and Henderson, R. J., Jr. Biochem. Med. 5, 212 (1971).
¹⁰Beutlcr, F., Pharinacol. Rev. 21, 73 (1969).
¹¹Huisman, T. H. J. and Schroeder, W. A., New Aspects of the Structure, Function and Synthesis of Hemoglobins, CRC Press, Cleveland, Ohio, 1971, p. 40.
¹²Mills, C. C., J. Amer. Sci. Affil. 20, 52 (1968).