THE
CLINICAL
IMPLICATIONS
OF
HEMOGLOBIN
STRUCTURE
E. Mead
Johnson
Award
Address,
October,
I 962
Park S. Gerald, M.D.
Department of Pediatrics, Harvard Medical School, and the Department of Medicine,
Chil4ren’s Hospital Medical Center, Boston, Massachusetts
This work was supported in part by a grant from the John A. Hartford Foundation and a PHS grant (11-4706) from the National Heart Institute.
ADDRESS: The Children’s Hospital Medical Center, 300 Longwood Avenue, Boston 15, Massachusetts.
PEDIATRICS, May 1963
780
I
FEEL DEEPLY HONORED to be named asone of the recipients of the 1962 Mead
J
ohnson Awards for research in pediatrics. I am grateful for this opportunity to ex-press my indebtedness and gratitude to the many who have aided me, and especially to my sponsor, Dr. Louis K. Diamond. His un-wavering confidence and unselfish support were major contributing factors toward the successful completion of these research ef-forts. Lack of time prevents me from giving an adequate list of those other teachers and associates who contributed significanfly toward my research efforts and training, but time can never be so short as to prevent me from mentioning my stimulating and ener-getic co-worker, Dr. Mary Efron. I have saved to the last, mention of my wife, as she has so often cheerfully been the vital silent partner of experiments that seemed always to last “just a little longer” than I expected.In order to make this a coherent presen-tation, I am going to dwell solely upon the “Hb M diseases.” This group of
hemoglo-binopathies is characterized by the
pres-ence of cyanosis in several generations of a given family, being transmitted as if deter-mined by the presence of a single abnormal gene (i.e., “dominant” inheritance).1 In the first family of this disorder that came to our attention, the cyanosis was an incidental finding in a child whose chief complaint
was an obscure peripheral neuritis. There
seemed to be little relation between the peripheral neuritis and the cyanosis, since the child’s brother, father, paternal aunt and other relatives also were cyanotic (Fig. 1), although lacking any neuritis.
The father gave us an interesting and, we later found, typical story of his own dis-ease. When he was a child, his parents were told he had a bad heart, for which restricted activity was advised. The con-tinued absence of any disability, however, caused his parents to ignore this advice, and he led a fully active childhood. In college
he participated in athletics without observ-ing any ill effects. Despite this, physicians repeatedly informed him that he was seri-ously ill. This caused many a frustration and annoyance, which reached a peak at the time he attempted to enlist in the Armed Forces. According to his tale, the examining physician merely glanced at him and peremptorily declared him unfit for
service. Fortunately, this man kept his own
and not his doctors’ counsel throughout all this-especially since at least one physician tried very hard to persuade him to consent to cardiac catheterization.
Examination of this man’s blood
elimi-nated the possibility that his cyanosis was secondary to anoxemia from cardiac or pul-monary disease, since the abnormal color of the blood was not altered by exposure to air. Hereditary methemoglobinemia due to an enzymatic defect, the next most likely possibility, could be easily ruled out by the negative results obtained with the usual spectroscopic test which, significantly, de-pends upon a change of color when cyanide is added to methemoglobin-containing
blood. Furthermore, ascorbic acid therapy, which has been reported to alleviate the cyanosis of methemoglobinemia, was unsuc-cessful in this patient.
possi-D CYANOTIC, BY HISTORY
D CYANOTIC, BY APPEARANcE 0 FEMALE
hility of an abnormal hemoglobin, since the
literature contained a report of a family in
whom a dominantly transmitted cyanosis
was thought to be caused by a defect in the globin portion of the hemoglobin.’ Electro-phoresis of the whole hemolyzate from our cyanotic man was used successfully to achieve a partial separation of an abnor-mally colored fraction from the normal
hemoglobin. With this to encourage us, we
were able to persist through the many trials necessary to discover a means of separating cleanly the normal from the abnormal
hemoglobin. The final method used
de-pended upon oxidizing the hemoglobin mixture to methemoglobin by adding potas-sium fernicyanide. It was found that after such treatment the abnormal methemoglo-bin of his blood readily separated from the normal methemoglobin fraction.’ It should be pointed out here that only hemoglobins of the Hb M group show this uniquely sue-cessful separation of the methomoglobin
forms.
Now that the abnormal hemoglobin
(des-ignated Hb or Hb M) could be
isolated, it was possible to study the prop-erties of the purified material. It was found that the Fib MB, which of course was abnor-mal in its electrophoretic properties, was
also abnormal in its spectroscopic behavior. Extensive spectroscopic studies of the iso-lated material led us to the venturesome
speculation that only part of the hemoglobin molecule was behaving peculiarly, while the remainder was acting in a normal fash-ion.3 (In the elaboration of this hypothesis, we benefited from the advice of our col-lahorator, Dr. Philip George.) In its final
form, the hypothesis pictured the four heme
groups of each molecule of Hb M as being
divisible into two heme groups with normal
reactivity, and two with abnormal
reac-tivity, the latter two being solely responsi-ble for the anomalous electrophoretic and
spectroscopic behavior of the molecule. Further, the heme groups with anomalous behavior were believed to have attained this state because the adjacent portion of the globin, the protein part of the molecule,
a
CYANOTIC, Hb M PRESENT 0 CYANOSIS ABSENT, BY HISTORY0 MALE
Fic. 1. Pedigree of initial family with Hb Detailed information is available for three
genera-tions only. The propositus is indicated by an
arrow.
was abnormal. Thus, the primary disorder
lay in the globin, and the alteration in heme
group reactivity was secondary. This con-ceptualization, of course, relied heavily upon the basic work of Ingram who had shown that several abnormal hemoglobins, which characterically are inherited as domi-nant conditions, had an alteration in the amino acid sequence of their globin, and that this alteration was confined to only two of the four parts, or protein chains, of the
The drawing of an analogy between Hb M80,0 and such abnormal hemoglobins
as Hb S or Hb C has its objections. This
(;EOGI(AIIII( I)IsTItIBtTIoN oF
un
1 l)IsE.sF:4. I’atie,ils . Patients
Location Location
(no.) (no.)
North America
Canada U.S.A.
South America
Asia
Japan
3
7
Europe England France
(;er,ii:i fly
Switzerland
Sweden
Israel
(;ret(e
3
3
Africa 0
782 HEMOGLOBIN STRUCTURE
* jTr(45te(l patients.’
Fn. 2. Starch block electrophoresis’ of the ox-i(liZ(’d hemolvzates (that is, the methemoglobins) at p11 7.0. The origin is indicated by the black lizw. Migration is toward the cathode. Top, nor-mal blood; middle, Fib Mskfl trait; bottom, Ill) trait. (Reprinted from Science by
I)erTflisSiOn).
glohin. It was natural then that some in-vestigators believed that any defects re-sponsible for a spectroscopically anomalous hemoglobin must reside in the heme groups,7 and not in the globin as we sug-gested.
Concurrent with this development of the-oretical aspects, a search was carried on for additional cases of cyanosis due to an
ab-normal hemoglobin. The dominant inheri-tance pattern typical of nearly all abnormal hemoglobins was chosen as the essential clue. Specimens were next obtained from a Canadian family with dominantly in-herited cyanosis who had been previously described in the literature as having con-genital methemoglobinemia.8 The same electrophoretic procedure was successfully used to isolate an electrophoretically and spectroscopically abnormal hemoglobin from these specimens (Fig. 2). While such success was gratifying support of the hy-pothesis, the situation rapidly became more complex when spectroscopic studies proved that this second hemoglobin was different from Hb vl Conditions rapidly became
worse ‘lien a third and then a fourth
spec-troscopically distinct variety of Hb I were discovered in two additional families with dominantly inherited cyanosis!’ Far from being the extremely rare disease it was
once thought to be, cyanosis due to
ab-normal hemoglobins (vhich are now cate-gorically known as the hemoglobins XI) has been encountered in iimny parts of the world (Table I). While not a common dis-ease, it would now seem to he a necessary part of the differential diagnosis of cyano-sis. Of particular interest is the absence of
any reported Hh NI trait in Negroes,
(IC-spite the prevalence of abnormal hemoglo-bins in this racial group.
The task of explaining how globin
alter-ations could produce these mans’ different varieties of spectroscopic abnormalities
was a challenging one. The methods for
demonstrating glohin defects had been ade-quately developed by ingram in his studies
of Hb S and Hh C.5 The same general ap-proach was used for the studs’ of the
hemo-globins M. The purified abnormal hemo-globins were digested by trypsin, and the peptides so produced were separated by
paper electrophoresis and/or paper chroma-tography. The separated peptides were then stained with reagents specific for certain amino acids (histidine, arginine, tyrosine, and tryptophan). The peptide patterns (des-ignated “fingerprints” by Ingram) were
ARflCLS
then compared to the peptide pattern ob-tamed with normal adult hemoglobin (Hb A). With four different examples of Hb M (obtained from unrelated individuals) four
different peptide patterns were obtained, each of which also differed from the pep-tide pattern of Hb A.’#{176}An example of one of these patterns is given in Figure 3. The
demonstration of an altered peptide pat-tern is evidence of an altered amino acid seiuence in the original globin and
con-firms the unique nature of each of these
hemoglobins. The hypothesis mentioned previously-that the basis for a spectro-scopically anomalous hemoglobin can reside in the globin portion-can now be said to be fact. This of course does not prove that all of the so-called hemoglobins M are the
result of globin defects, although it seems likely that most will follow this rule.
Demonstration of a globin defect did not by itself answer the question of how this defect could produce an anomaly of
spec-troscopic behavior. At this point, our col-laborator, Dr. Philip George, furnished the invaluable suggestion that the altered amino acid sequence of the globin, indicated by
the aberrant peptide patterns, might result
in a “new” amino acid being near the heme
group. This “new” amino acid could then react with the heme group and affect its spectroscopic properties. To determine that one part of the molecule is “near” a heme
group, however, requires an intimate knowl-edge of the three-dimensional structure of hemoglobin. While such direct information
is lacking for human hemoglobin, a model
for horse hemoglobin has been published by Perutz and his co-workers.’1 One of the cur-rent “break throughs” in the study of hemo-globin is the discovery that the four protein
chams of horse hemoglobin are similar to one another in their three dimensional structure. In addition, there are many simi-larities between the amino acid sequence of the chains of human hemoglobin and the
chains of horse and other animal
hemoglo-213 This has led to the concept of
“homology” which embodies the belief that all vertebrate hemoglobins differ from one
20/3 20a 21/3 21a
::5jp$J9
HFi#{174}
FIG. 3. Schematic representation of the one-di-mensional electrophoretic patterns (pFl 6.5) of
tryptic digests of Hb A and Hb M, after staining with ninhydrin.’#{176} An abnormal peptide, the an:s-logue of 21 1, is present in the Ms pattern just in advance of the 20 a band. (Reprinted from
Proc. Nat. Acad. Sci. by permission).
another largely by isolated replacements of one amino acid for another in the otherwise unaltered amino acid sequence. Homology leads to the expectation that human hemo-globin will have very nearly the same three dimensional structure as horse hemoglobin. This permits us to depict each of the four
heme groups of human hemoglobin as being enclosed within a fold of one of the
four protein chains of the molecule- one heme group per chain (Fig. 4). It is evident
a 58, 63
a 62,
67
FIG. 4. Schematic representation of the fold of a
hemoglobin chain (symbolized by the heavy line) with its contained heme group. The heme group is pictured as a disc seen from the side. The iron atom is located in the center of the heme disc.
The amino acids whose side groups project from the axis of the protein chain toward the heme iron are indicated (His-histidine, Val-valine). The same amino acids are found in both the alpha and beta chains, at the points in the chains
specified. The numbers are determined by
TABLE III
VISIBLE ABsom’TIoN SI’Fc’ra& FOli (YA NM ETII EM(
)-GLOBIN I)EI4IVATIVE OF S::,isi \SIUETIKs
OF IIB M
Abnormal (
‘yaninethemo-Hb 11 Type Chain glDbin Spectrnii
784 HEMOGLOBIN STRUCTURE
‘I’ABLE IL
A uINO A(1 I) SEQUENCE ALTERATIONS IN I)t
FFEIt-ENT VARIETIES OF HB M
A,nini Arid “I)ifferenee’ Jib M Type
from JJb .4
Hb a25 (a58, his-tyr)
Hb MSa,katoon a2$2* ($63, his-*tyr)
HI) MMIL,k,_1 #{163}Y2$2*(fl67, val-+glu)
Hb Miate a2*132 (a6-9O, ?-*tyr) Hh MMilwaukee_2 prol)ably a22’
* Indicates abnormal chain. T.e symbols in
pareil-thesis refer to the residue which is altered, with the
arrow pointing towar1 the residue found in the Hb M.
from the horse hemoglobin model that the molecule consists of two identical halves, each half containing two different protein chains, which are now known as the alpha and beta chains. The whole molecule with
its four chains can then be written as
allorse Horse Human hemoglobin then is 1Iunan 1Iuman or, as it is more commonly
written, simply a2 On the basis of
ho-mology, Kendrew has predicted which amino acids in the alpha and beta chains of human hemoglobin should be in immediate
proximity to the heme group (Fig. 4).14
When the abnormal peptides of the Hb M fingerprints were analyzed for their amino acid content, in the three cases where the analysis could be accurately
per-formed, one of the normal amino acids was missing in each instance and a “new” amino acid was present (Table II). In all three hemoblobins the amino acid found to be missing was one of those expected to be in the immediate vicinity of the heme group. It is therefore considered probable that the
“new” amino acids replace the missing nor-mal amino acids, and hence also are in the immediate vicinity of the heme group.’#{176} They are thus able to influence the heme group of their particular protein chain, and only that heme group, as demanded by the hypothesis stated earlier.
It is now possible to offer an explanation for some of the clinical aspects of the Hb Nil diseases. The cyanosis apears to be due to the unusual stability of the methemoglobin
M,ton a Abnormal
Miwate a Abtiormal
MSaskatoon Normal
MMi1waukee1 l Normal
MMiIwaukee2 #{237}9(?) Normal
form of the hemoglobins M. This is be-cause each of the “new” amino acids is a potential negatively charged group which can bind or internally complex with the oxidized iron atom of methemoglobin. The resulting complex would be expected to be unusually resistant to the reducing action of the enzymes in the red cell. The complex might also in some cases be so stable that the ferric atom would be unable to react with outside agents such as cyanide, which normally reacts with methemoglobin to form cyanmethemoglobin. Failure to react with cyanide should also lead to an abnor-mal cyanmethemoglobin absorption curve.
In Table III the cyanmethemoglobin spectra for the several chemically studied
varieties of Hb M are classified as normal or abnormal. As is indicated, Hb MBO,t0fl has an abnormal cyanmethemoglobin spec-trum which, according to the foregoing,
in-dicates that the hemes of the abnormal chains are unable to react with cyanide. This is consistent with the finding that the spectroscopic test for methemoglobin, which depends on this reaction, was nega-tive in this patient. In Hb MSaSkatOOrI (Hb
M5), on the other hand, the cyanmethemo-globin curve is normal and the spectro-scopic test for methemoglobin in the blood
was positive in this patient.
con-vinced, of the need for a thorough knowl-edge of the three dimensional structure of
proteins in general in order to understand
their several roles in physiologic and patho-logic states. It is only by this kind of an approach that a rational attack on such problems as, for instance, the sickling of Hb S and the treatment of sickle cell anemia can be attempted. The promise of this area of investigation seems bright indeed.
REFERENCES
1. Gerald, P. 5. : The hereditary methemoglo-binemias, in The Metabolic Basis of In-herited Disease; edited by Stanbury, Wyn-gaarden, and Fredrickson. New York, Mc-Graw-Hull, 1960.
2. ll#{246}rlein, H., and Weber, C. : Uber chronische famili#{228}re Methamoglobinamie und eine neue Modifikation des Methamoglobins. Deutsche
Med. Wschr., 73:476, 1948.
:3 Gerald, P. : The electrophoretic and
spectro-scopic characterization of Hgb M. Blood,
13:936, 1958.
4. Gerald, P., and Ceorge, P. : Second
spectro-scopically abnormal methemoglobin
asso-ciated with hereditary cyanosis. Science,
129:393, 1959.
5. Ingram, V. NI. : Chemistry of the abnormal human haemoglobins. Brit. Med. Bull., 15: 27, 1959.
6. Lemberg, R., and Legge, J. W. : Hematin Compounds and Bile Pigments. New York, Interscience, 1949, p. 309.
7. Beaven, G. H., and Gratzvr, W. B. : A critical review of human haemoglohin variants. J.
Clin. Path., 12:1, 1959.
8. Baltzan, D., and Sugarman, ii. : Hereditary
cyanosis. Canad. Med. Ass. J., 62:348,
1950.
9. Specimens from most of the patients referred to in this table have been examined in the
author’s laboratory. In a few instances personal communication or published
ac-counts have been the source of informa-tion.
10. Gerald, P. 5., and Efron, M. L. : Chemical
studies of several varieties of Hb M. Proc.
Nat. Acad. Sci., 47: 1758, 1961.
1 1. Perutz, M. F., et a!.: Structure of hemoglobin.
Nature, 185:416, 1960.
12. Braunitzer, C., and Matsuda, C. : Die Analyse
der tryptischen Peptide des Pferdeh#{228}mo-globins. Z. Physiol. Chem., 325:91, 1961. 13. Diamond, J. M., and Braunitzer, C. : a Chain
of rabbit haemoglobin. Nature, 194:1287, 1962.
14. Watson, H. C., and Kendrew, J. C. : Compari-son between the amino-acid sequences of sperm whale myoglobin and of human haemoglobin. Nature, 190:670, 1961.
15. Gerald, P. S., and Efron, M. L. : Unpublished
data.
A PHILOSOPHY OF INFANT FEEDING, by
Simon S. Levin, Springfield, Ill., Charles C Thomas, 1962, 175 pp., $7.00.
This is a delightful book.
In the author’s introduction it is stated:
“There are so many books on the feeding of
infants. Whatever its faults, at least this little
book is different.” This book is especially
dif-ferent in that infant feeding is covered in a
very painless fashion with a minimum of tables and figures. The writing is excellent, and one
hardly realizes that he is reading a textbook
of fact.
The author uses a paleocentric approach to infant feeding. In his evolutionary study he
finds for example, “In modern cultures it is
no longer a case of survival of the breast
fit-test.” In addition to many hon mots the book
is replete with helpful practical advice.
The special value of breast feeding is well explained. The composition of milk is listed
casually. The information on feeding of
pre-matures is helpful, and the discussion on de-velopment of infant’s enzymes is up to date for the major systems.
One might take issue with the rather cava-her attitude of the author towards diarrhea, and one might also question the author’s
state-ment regarding the salt content of milk. A bibliography would be desirable. These are, however, negligible objections. The book is very well written; the format is excellent and
easy to read.
This book should be read by medical
stu-dents, pediatricians, general practitioners,
flu-tritionists and ans’ one who cares for infants.
