A new abnormal human hemoglobin Hb: Zurich

CLINICA

A NEW

CHIMICA

ABNORMAL

ACTA

HUMAN

347

HEMOGLOBIN

Hb: ZURICH TITUS

Departments

H. J. HUISMAN,

of Biochemistry

BEl\jSETT

HORTOX,

KLAUS

Universitdts-Kindevklinik,

T. BRIDGES

BETKE

Freiburg in Breisgau (Germany)

ANU WALTER

H. HITZIG

Universitiits-Kinderklinik, (Received

In recent

MERCER

and Pathology, Medical College of Georgia, Augusta, Ga. (U.S.A .)

October

years the many different

have evoked much interest studied intensively because considered to be mutations

and study.

Zurich (Switzerland) moth, 1960)

types of hemoglobin Their medical

found in human beings

and genetic

aspects have been

several of the abnormal hemoglobin types, which are in a Mendelian gene, are associated with a definite

pathological

entity. Due to the development of new techniques and in particular “fingerprint” technique1 new possibilities for a study of the biochemical differences have become available. Several abnormal hemoglobins have been studied in this way, and, surprisingly, their structural changes as compared with normal adult hemoglobin are small and mostly confined to an exchange of one amino acid INGRAM’S

for another in one of the two types of polypeptide chains of the protein, Small differences have been described for the abnormal hemoglobins S, C and E2, the D haemoglobin9, Hb-G4p5 and H-16. For most abnormal Hb-types the abnormality is located in the p-chain, but abnormalities of the a-chain (for example, Hb-I) have been discovered. Next to these abnormalities other forms of human hemoglobin have been described in which the a-chain is lacking, for example, Hb-H, Bart’s Hb, HbAugusta I, Hb-Augusta II, which were found to consist solely of the b-chain of normal adult Hb (Hb-H)‘, the y-chain of fetal hemoglobin (Bart’s Hb)s, the abnormal p-chain of sickle cell Hb (Augusta I)9 and the abnormal/?-chain of Hb*C (Augusta II)lO. The present paper is concerned with an abnormal form of human hemoglobin discovered in a white family of purely Swiss descent. Structural studies have revealed some different abnormalities located in the p-chain, while the cc-chain of the abnormal component was identical to that of normal Hb-A. Since no definite conclusion concerning its identity with one of the known abnormal Hb-types could be drawn, the discovery of a new abnormal type is most likely and the tentative name “Hb-Zurich” is proposed. MATERIAL

AND

METHODS

The family Sch. of pure Swiss extraction came under investigation because of severe Heinz body anemia in a female infant then 3 years old. Starch electrophoresis of the hemoglobin of this patient, carried out by one of us (K.B.), revealed the presence of a slow moving hemoglobin component, which was also detectable in the blood of the father; the hemoglobin pattern of the mother was normal. Hemoglobin samples from both the father (Mr. Sch.) and his daughter (Child Sch.) were available for the study Clin. Chim. Acta, 6 (1961)

347-355

348

T. H. J. HUISMANet Ul.

presented in this paper, which is mainly dealing with the chemical characterization of the abnormal hemoglobin component. The clinical and genetic aspects of the abnormality will be described separately ll. The component was also found to be present in the blood of many other relatives belonging to four generation+. Electrophoretic studies were performed using paper electrophoresis at PH 8.6 as well as at pH 6.5, starch gel electrophoresis at pH 8.0 following the technique described by SMITHIES~~ I2 and starch block electrophoresis according to the procedure described by GERALDANDDIAMOND13.The spectrophotometric alkali denaturation procedure by JONXIS AND VISSER’~was used for the demonstration of alkali resistant hemoglobin. Chromatographic analyses were carried out by the Amberlite IRC-jo chromatographic procedure previously describedlsj 16,and by carboxymethyl cellulose chromatography17> ln. With this last procedure quantitative data were obtained. The solubility of the reduced hemoglobin was studied with the method described by ITANO’~. The abnormal component was isolated and purified with the carboxymethylcellulose chromatographic procedure using 50 cm x 0.9 cm columns of CM-cellulose, 0.01 M phosphate buffers and the pH gradient described earlier’*. The purity of the abnormal component was tested by starch gel electrophoresis. Part of the isolated abnormal hemoglobin was studied by the “fingerprint” technique developed by INGRAM’.The remaining Hb was converted to globin and separated into the constituent polypeptide chains by the chromatographic technique given by WILSON AND SMITH~O and also used in other [email protected] isolated polypeptide chains were digested with trypsin and the resulting mixtures of peptides studied for possible structural abnormalities following the procedure described by INGRAMAND HUNTER. The color reactions specific for various amino acids (ninhydrin, histidine, arginine, tyrosine and tryptophane) were carried out as described in the handbook of BLOCK,DURRUMAND ZWEIG~~.Hemoglobin samples obtained from two of the authors were used as normal controls. The possibility of an abnormal u-chain being present in this hemoglobin was VINOGR~ZD also studied by the “hybridization procedure” described by SCHROEDER, and coworkers7yz3, which was also used in a previous study24. Equal amounts of the pure abnormal Hb-Zurich and Hb-types with known /?-chain abnormalities (Hb-C and Hb-B,)24 were mixed and dialyzed at pH 4.7 7~23for 48 h at 4” followed by dialysis at PH 8.0 for 48 h. The resulting mixtures of hemoglobin types were studied using the CM-cellulose chromatographic procedure previously mentioned. RESULTS The characterization

of the abnormal

component

With paper electrophoresis both at pH 8.6 and at pH 6.5 the presence of an abnormal hemoglobin component in the blood of both cases was detectable (Fig. I). At PH 8.6 a faint tail was visible, which moved behind the normal Hb-A but faster than the abnormal Hb-S. At pH 6.5 this component is not separated from Hb-A but is moving slightly faster than Hb-A and distinctly slower than Hb-S. The amount of the abnormal Hb seems to be higher in the blood of Mr. Sch. than in that of his daughter. With starch electrophoresis as well as with starch gel electrophoresis the separa-

A NEW

Fig. 1. Paper electrophoretic

ABNORMAL

HUMAN

HEMOGLOBIN

patterns of the hemoglobins of Mr. Sch. and his daughter.

Fig. 2. The electrophoretic patterns (by starch electrophoresis and by starch gel electrophoresis) of the hemoglobins of Mr. Sch., his daughter and a known case of sickle cell trait.

Fig. 3. The separations of the hemoglobins of child Sch. and of a case heterozygous for the Hb-S by CM-cellulose chromatography.

C&n. Chim. Acta, 6 (1961) 347-355

T.

350

H.

J. HUISMAN

t%!d.

tion of the abnormal component and normal Hb-A is complete (Fig. 2). The starch electrophoretic pattern showed Hb-Zurich moving behind Hb-A and slightly faster than Hb-S. Small amounts of the minor component Hb-A, were also detectable in both hemoglobin samples. Starch gel electrophoresis revealed the Hb-Zurich to have a mobility similar to that of Hb-S. Except for small amounts of Hb-A, no other Hb-components were detectable. The two electrophoretic patterns showed again that the abnormal component was present in higher quantities in the blood of the father than in the blood of the 3 years old child. The amount of alkali resistant Hb present in both samples was low; the value found for the blood of the father was within the normal limits (less than z%), while in the child a definite increased amount was detectable (4%). Chromatographic the abnormal When mixed clearly

studies using the Amberlite

component with Hb-S,

separated

from the normal

method

have shown that

adult hemoglobin. TABLE

THE QUANTITATI”E

IRC-50

moved slower than Hb-A but definitely faster than Hb-S. the Hb-Zurich moved slightly faster than Hb-S and was

I

AMOrJNTS OF THE DIFFERENT HEMOGl,OBIN FRACTIONS PRESENT IN THE TWO CASESOFFAMILY Sch. v1

V2

241

d 0

1;

Zurich

Sch.

0.2

0.1

12.3

56.6

0.9

28.4

Child Sch. ____..~~ ~

0.4

0.4

9.5

60.4

5.4

22.3

Father

TABLE SOLUBILITIES

OF

REDUCED

HEMOGLOBINS

IN

_

--

I.5 1.6

II

CONCENTRATED

2.24 M Phosphate Hb-A

A2

PHOSPHATE 2.56

BUFFERS

OFPH

6.5

M Phosphate

95

29

Sickle cell trait

45

Mr. Sch.

92

73 26

Child Sch.

91

32

Similar results were obtained when the two hemoglobin samples were studied by the CM-cellulose chromatographic technique. In Fig. 3 the result obtained for the hemoglobin of the daughter is compared with that of a known case of sickle cell were present and the abnormal component was trait. Different normal fractionP completely separated. Its mobility was found to be slightly greater than that of Hb-S resulting in a complete separation of Hb-Zurich and the minor Hb-A, component. A small amount of Hb-F in the hemoglobin of child Sch. was also detectable, which corresponded with the increased percentage of alkali resistant hemoglobin detectable with the spectrophotometric alkali denaturation procedure. Table I presents the quantitative results obtained by CM-cellulose chromatography. The amounts of Hb-Zurich present in the blood of the father was higher than of the child, which corresponded with the results obtained by electrophoretic procedures. The amounts of Hb-A, were low but still in the normal range. The percentage of Hb-F in the blood of the child corresponded reasonable well with that determined by the alkali denaturation technique. The difference between Hb-Zurich and sickle cell Hb was also proven by comClin. Chim. Acta, 6 (x961) 347-35.5

A

NEW

ABNORMAL

HUMAN

HEMOGLOBIN

351

paring the solubilities of the reduced hemoglobins of Mr. Sch. and of his daughter with those of a normal individual and of a patient with sickle cell trait. From the results given in Table II it is evident solubility as normal Hb-A. STRUCTURAL

The purity

STUDIES

of the abnormal

that the Hb-“Zurich”

ON THE

ISOLATED

Hb-component,

ABNORMAL

showed

the same high

HEMOGLOBIN

isolated by CM-cellulose chromatog-

raphy, was tested by starch gel electrophoresis and by rechromatography on a CM-cellulose column. No significant amount of normal Hb-A nor of any other Hb-component was detectable. The pure Hb-Zurich was first studied for the presence of an abnormal cc-chain by mixing with abnormal hemoglobins with known p-chain abnormality. The ABNORMAL

Hb

Hb-A

Fig. 4, Tracings of fingerprints of the abnormal Hb-Zurich and of Hb-A; for explanation see text. Abnormal Hb: tryptophan, 12, 16, 8 ?; histidine, I, 2, 4, 9, IO, 15, 16, 17?. 20, 23; arginine, 16, 18, 23, 26, A, 17 ?; tyrosine, 2, IO, 15, 16, 23, 24 or 25 faint, 4? Hb-i\: tryptophan, 12, 16; histidine, I, 2, 4, 9, IO, 15, 16, IT?, 20, 21, 23; arginine, 16, 18, 23, 26, 17?; tyrosine, 2, IO, 15, 16, 23, zsfaint, 4?

mixtures were dialysed at the dissociating pH of 4.7 for 48 h at 4” followed by dialysis at PH 8.0 for 48 h and finally analyzed by CM-cellulose chromatography. During these reversible dissociation and recombination experiments the formation of “hybrids” may be expected only when abnormal a-chains combine with normal or abnormal B-chains. The hybridization of Hb-Zurich was carried out with the abnormal Hb-C and separately with the abnormal Hb-B,24 both known to be composed of normal a-chains and abnormal p-chains or &chaims. The chromatographic analyses yielded only the two hemoglobin types originally present in the mixtures, indicating a close similarity of the a-chains of Hb-Zurich and of the Hb-C and Hb-B,. The structural abnormalities of the abnormal Hb component were studied by comparing the “fingerprints” of the tryptic digest of Hb-Zurich with that of normal adult hemoglobin. The results are presented in Fig. 4. These differences were detectable: (a) ninhydrin stain: peptide No. 21 is absent in Hb-Zurich; three new peptides (labeled A, B and C) were detectable, located between peptides 19 and 22 (peptide A), between peptides 20 and 22 (peptide B), while peptide C is located below peptide No. 22 and on the negative side of the missing peptide 21; peptide 26 of the abnormal Hb is less negative than the corresponding peptide of Hb-A. (b) Histidine Clin.

Chim.

Acta,

6 (1961)

34,-355

T. H. J. HUISMAN

352

et d.

stain: the absence of peptide 21 in Hb-Zurich was confirmed; no histidine was detectable in the new peptides A, B and C. (c) Arginine stain: Peptide A contained arginine; arginine was present in the abnormal peptide 26, confirming the abnormal position of this peptide in Hb-Zurich. (d) Tryptophane and tyrosine stains: no abnormalities; these amino acids were absent in the peptides A, B and C. The globin derived from Hb-Zurich was converted into the cc-and p-chains

fichoin

o(

Fig. 5. Schematic Hb-Zurich and of 1+2, 4, 23, IT?); (also I + 2, 4, 23, 1 + 2, 9, 10, 16?,

chain

ABNORMAL

ABNORMAL

Hb

f3

chain

by

tib-A

Hb

representation of the fingerprints of the cc-chain and b-chains of the abnormal Hb-A; for explanation see text. p-chain abnormal Hb: histidine, 15, 16, (also arginine, 16, 26, A, IT?, (also 18, 23). p-chain Hb-A: histidine, 15, 16, 20, 21, 17 ?); arginine, 16, 26, 17 ?, (also 18, 23). m-chain abnormal Hb: histidine, 23, 20; arginine, 23, 18, 16?. cc-chain Hb-A: histidine, 23, I + 2, 9, IO, 16?, 20; arginine, 23, 18, 16?

applying Amberlite IRC-50 chromatography at PH I.9 with a 2-S M urea gradient. The urea concentrations, at which the a- and @-chains of globin Zurich were eluted, were similar to those found for Hb-A (a-chain Hb-Zurich 5.9 M; a-Hb A 5.8 & 0.1 M; /?-chain Hb Zurich 7.1 M; ,&Hb-A 6.9 5 0.1 M). The peptide mixtures obtained after tryptic digestion of the a- and @chains of the abnormal hemoglobin were studied in the same way as the total digest of HbZurich. The resulting “fingerprints” were compared with those of the corresponding peptide chains of normal Hb-A. The results presented in Fig. 5 showed a close identity of the a-chains of the abnormal Hb and normal Hb-A. The B-chain of HbZurich, however, is different from that of Hb-A, the differences being almost identical with those described for the “fingerprint” of the tryptic digest of the total abnormal

A NEW

Hb. It seems therefore

ABNORMAL

HUMAN

that the abnormalities-the

HEMOGLOBIN

353

presence of the three new peptides,

the absence of peptide 21 and the abnormal peptide 26-are located in the p-chain. With the arginine stain the presence of this amino acid in peptide A was confirmed, as was the abnormal position of peptide 26. Application of the histidine stain revealed one extra abnormality: next to the absence of the histidine containing peptide 21, no histidine was present in peptide 20 of the B-chain of Hb-Zurich in contrast to the /?-chain of Hb-A. Peptide 20 of the a-chain of Hb-Zurich, on the contrary did contain histidine, abnormal

which explains the positive reaction of peptide Hb with this specific amino acid reagent,

20

of the digest of the total

DISCUSSION

The abnormal hemoglobin component found in this family is characterized by the following properties: (a) its electrophoretic mobility is intermediate between Hb-A and Hb-S. Although the results of the electrophoretic patterns obtained with different techniques (paper-, starch- and starch gel electrophoresis) were not completely comparable, it seems likely that the mobility is closer to that of Hb-S than to that of Hb-A. (b) In Amberlite IRC-50 chromatography its mobility is slightly faster than that of Hb-S; similar results were obtained in CM-cellulose chromatography. (c) The abnormal hemoglobin is not resistant to alkali. (d) The solubility of the abnormal component (in reduced form) is similar to that found for Hb-A. (e) The Hb-Zurich possesses some structural abnormalities, located in the p-chain of the protein. Like other abnormal hemoglobins, the presence of Hb-Zurich seems to be determined by a single gene which when heterozygous results in about 30 per cent of the hemoglobin being abnormalll. Several known abnormal Hb-components possess comparable electrophoretic mobilities, such as Hb-GZ5, Hb-P26-2B, Hb-QZ9, Hb-Lepore30, Hb-Alexandra319 32, Hb-Stanleyville 133, Hb-Stanleyville II33 and Hb-Cyprus 134, but many of these abnormal hemoglobins show other properties which make them different from HbZurich. A possible identity of Hb-Zurich with most of these abnormal Hb’s can be excluded

for the following

reasons:

(a) Hb-Cyprus

and Hb-Alexandra,

most likely

abnormal fetal hemoglobins, are more or less resistant to alkali. (b) Stanleyville I and Stanleyville II, both found in Negroes, possess different chromatographic properties (Amberlite IRC-50) : Stanleyville I cannot be separated from Hb-S, and Hb-Stanleyville II shows a slower mobility than Hb-S. (c) Although most properties of HbZurich and Hb-Lepore are similar, the high amounts of the abnormal component found in our cases (22 and 28%), while the Hb-Lepore is present only for about IO%~~, make an identity of the two abnormal hemoglobins not likely. (d) the Hb’s P and Q can also be excluded since Hb-P shows a similar mobility in Amberlite IRC-50 chromatography as Hb-A, while Hb-Q possesses a slower mobility than Hb-S using the same chromatographic procedure. (e) Hb-G, also found in Negroes, is probably also different from Hb-Zurich due to small differences in chromatographic mobilities. It seems, therefore, that the abnormal component described in this paper is most likely not identical with any of the known abnormal Hb-types. A definite statement, however, cannot be made, since the abnormal components mentioned above were not available for the purpose of comparison. The results

obtained

by studying

the possible

structural

differences

Clin. Chim.

Acta,

between

6 (x96x) 347-355

T. H. J. HuISM.~N

354 the abnormal

hemoglobin

and normal

et al.

Hb-A have

shown, that

some abnormalities

are present in Hb-Zurich, which are located in the B-chain of the protein. One peptide (No. 21) was found to be absent while at least two peptides (20 and 26) were different in structure, since peptide 20 of the P-chain of Hb-Zurich did not contain any histidine and peptide 26 (containing arginine) had a different electrophoretic mobility. Moreover three new peptides (peptides A, B and C) were detectable in the tryptic digest of the p-chain of Hb-Zurich, one of them (peptide A) containing arginine. The possibility also exists that peptide 24 of the p-chain of Hb-Zurich is different from the corresponding peptide of the P-chain of Hb-A, since the chromatographic mobilities of the two spots were slightly different. There has been expressed the opinion that certain abnormal hemoglobins (Hb-S and Hb-C for instance) differ from normal adult hemoglobins in only one amino acid residue located in one of the types of polypeptide chains. This opinion was based on the fact that their “fingerprints” appeared identical in all respects except for one peptide, the amino acid sequences of which have been found to differ by only one amino acid2. In this respect our results obtained by studying the structural differences between Hb-Zurich and normal Hb-A bring up some interesting points. First the fingerprints of Hb-Zurich showed different abnormalities resulting for instance in the absence of a normally occurring peptide, the presence of some new peptides and differences in mobilities of some other peptides present in the tryptic digests of the total hemoglobin and of its P-chain. Secondly, it will be clear that the abnormality in peptide 20 of the p-chain (i.e. the absence of histidine) could be detected only by studying the fingerprint of the isolated p-chain, since peptide 20 present in the a-chain of Hb-Zurich did contain histidine and posessed similar electrophoretic and chromotographic properties as found for peptide 20 of the p-chain. This should lead us to conclude that when two different hemoglobin preparations give similar spots in the “fingerprint”, they are not necessarily identical. This leads to the premise that definite proof of identity or non-identity of two hemoglobins must await elucidation of the entire amino acid sequence of both hemoglobins. Our results support the idea, that differences between hemoglobin types with close genetic relationships of the genes responsible for their formation are not necessarily limited to one change in their polypeptide structures. ACKNOWLEDGEMENTS The authors are highly indebted to Miss J. B. SEBENS for her capable technical assistance. The authors are also obliged to Dr. H. LEHMANN, London, and Dr. P. S. GERALD, Boston, for their interest in these cases. M. T. BRIDGES was holding a student fellowship offered by the Cancer Teaching Program of the Medical College of Georgia, Augusta (U.S.A.). The authors are also indebted to the Department of Medical Illustration (Med. College of Georgia) for their assistance in preparing the figures. The investigations were supported by a grant of the U.S. Public Health ServiceNo. H-5168.

I. An abnormal slow moving hemoglobin, found in a white Swiss Family, is described. The abnormal hemoglobin is characterized by its electrophoretic and

A NEW

chromatographic hemoglobin

types.

mobilities.

ABNORMAL

HUMAN

It was found to be different

The component

355

HEMOGLOBIN

is alkali non-resistant

from the knownabnormal and, in the reduced

state,

possesses the same solubility in concentrated phosphate solutions as found for Hb-A. 2. The amounts of the abnormal component present in the blood of the father and his daughter were 28.4 and 22.3% respectively. Similar quantities were present in other members of the family of the father. 3. Hybridization experiments revealed no abnormality in the a-chain of the protein. 4. The fingerprints of the digests of the total hemoglobin and of its p-chain have shown multiple differences as compared with normal Hb-A. The fingerprints of the a-chains

of the abnormal

component

and of Hb-A were identical.

plications of these results are discussed. 5. The tentative name Hb-“Zurich”

The genetic

im-

is proposed.

REFERENCES 1 V. M. INGRAM, Biochim. Biophys. Acta, 28 (1958) 539. 2 V. M. INGRAM, Brit. Med. J., 15 (1959) 27. 3 S. BENZER, V. M. INGRAM AND H. LEHMANN, Nature, 182 (1958) 852. 4 R. L. HILL AND H. C. SCIIWARTZ, Nature, 184 (1959) 641. 5 R. L. HILL, R. T. SWENSON AND H. C. SCHWARTZ, Federation Proc., 19 (1960) 77. 6 M. MURAYAMA, Federation Proc., 19 (1960) 78. 7 R. T. JONES, W. A. SCHROEDER, J. E. BALOG AND J. R. VINOGRAD, J. .4m. Chem. Sot., 81(1959) 3171. 8 J. A. HUNT AND H. LEHMANN, Nature, 184 (1959) 872. 8 T. H. J. HUISMAN, CZin. Chim. Acta, 5 (1960) 709. 10 T. H. J. HUISMAN, Nature, 188 (1960) 589. 11W. H. HITZIG, P. G. FRICK, K. BETKE AND T. H. J. HUISMAN,

Helv. Paediatr. Acta, 15 (1960)

499. I2 0. SMITHIES, B&hem. J., 71 (1959) 585. I3 P. S. GERALD AND L. K. DIAMOND, Blood, IZ (1957) 61. 14 J. H. P. JONXIS AND H. K. A. VISSER, A.M.A. J. Diseases Children, 92 (1956) 580. I5 T. H. J, HUISMAN AND H. K. PRINS, J. Lab. C&z. Med., 46 (1955) 255. I6 T. H. J. HUISMAN AND H. K. PRINS, CZin. Chim. Acta, z (1957) 307. 17 T. H. J. HUISMAN, C. A. MARTIS AND A. DOZY, J. Lab. Cl&. Med., 52 (1958) 312. 18 T. H. J. HUISMAN AND C. A. MEYERING, CZin. Chim. Acta, 5 (1960) 105. IQ H. A. ITANO, Arch. Biochem. Biophys., 47 (1953) 148. 20 S. WILSON AND D. B. SMITH, Can. J. Biochem. Physiol., 37 (1959) 405. 21 J. A. HUNT, Nature, 183 (1959) 1373. 22 R. J. BLOCK, E. L. DTJRRUMAND G. ZWEIG, Paper Chromatography and Paper Electrophoresis, Academic Press, New York, 1958. 23 J. R. VINOGRAD, W. D. HUTCHINSON AND W. A. SCHROEDER, J. Am. Chem. Sot., Sr(Ig5g) 3168. 24 T. H. J. HUISMAN, B. HORTON AND T. B. SEBENS, Nature, in the press. 26 G. M. EDINGTON, H. LEHMANN AND R. SCHNEIDER, Nature, 175 (1955) 850. 26 R. SCHNEIDER, Nature, 180 (1957) 1486. 27 R. SCHNEIDER, Nature, 182 (1958) 322. 28 H. LEHMANN, Nature, 184 (1959) 1133. 29 T. VELLA, R. C. WELLS, J. A. M. AGER AND H. LEHMANN, Brit. Med. J., (1958 I) 752. 30 P. S. GERALD AND L. K. DIAMOND, Blood, 12 (1957) 61. 31 P. FESSAS, N. MASTROVALOS AND G. FOSTIROPOULOS, Nature, 183 (1959) 30. 32 F. VELLA, J. A. M. AGER AND H. LEHMANN, Nature, 183 (1959) 32. 33 I’. DHERTE, J. VANDEPITTE, J. A. M. AGER AND H. LEHMANN, Brit. Med. J., (1959 II) 282. 34 J. E. O’N. GILLESPIE, J. C. WHITE, M. J. ELLIS, G. H. BEBVEN, W. B. GRATZER, E. M. SHOOTER AND R. M. E. PARKHOUSE, Nature, 184 (1959) 1876.