Electron microscopic observations on human haemoglobins

Experimenfal

Cell Research

ELECTRON

D.N. Biophysics

MISRA, Division,

N.N. Saha

34,

325-332

(1961)

325

MICROSCOPIC OBSERVATIONS HUMAN HAEMOGLOBINS DAS In&tare School

GIJPTA,

A. B.

of Nuclear of Tropical

Physics, Medicine,

Received

May

SANYAL Calcu#~, Calcutta,

and

J. B.

ON

CHATTERJEA

and rhe Haernatology India

Department,

15, 1963

IN

some previous publications, preliminary results of the electron microscopic studies of human haemoglobins were reported [3, 91. The usual technique of shadowing the macromolecules with platinum vapour was utilised in those investigations and the shadowed micrographs gave definite evidence of substructures on the molecules. However, with the shadowing technique, the structure of the supporting film was very prominent causing confusion and it was also not possible to obtain accurate measurement of the dimensions of the macromolecules due to the uncertainty in the amount of metal vapour deposited on them. The present paper contains an account of electron microscopic investigations on human haemoglobin molecules, carried out with the negative staining technique. With the help of this method it has been possible to obtain micrographs with much greater resolution of the structural details than those obtained previously. Close inspection of the micrographs revealed many interesting features of the molecules which have not been observed before. Almost the complete structure of horse haemoglobin molecule has been determined by Bragg, Perutz and their collaborators from X-ray diffraction analysis [2, 4, 81. From the structural similarity between the polypeptide chain folds of sperm-whale and seal myoglobin on the one hand and of hor.se and bovine haemoglobins on the other, these authors concluded that all myoglobins and haemoglobins of vertebrates resemble closely. If it is true that the structures of horse and human haemoglobins are similar, one can make a fruitful comparison between the present electron microscopic findings on human haemoglobins and the deductions from S-ray diffraction for horse haemoglobin molecule. The X-ray diffraction studies show that the horse haemoglobin molecule is spheroidal in shape with the approximate dimensions: length 64 A, width 55 A and a height 50 di along a-, c- and b-axis respectively and that it consists of four subunits (two a chains and two p chains) in a tetrahedral 22

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641801

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D. N. Misra

326

et al.

array. The molecule contains two pseudo-dyads one of which is formed by pairing of the two similarly folded a chains and the other by pairing of the /3 chains. These two lie approximately at right angles to each other and to the true dyad. It has a dimple where the a chains meet and a larger hollow where

Fig. l.-The globins.

ultraviolet

absorption

curves

of the

harmo-

the /I chains meet, the two depressions being connected by a hole along the b-axis. These deductions from S-ray diffraction studies on the structure of horse haemoglobin have been very carefully compared with the electron microscopic observations on stained human haemoglobin molecules in the present paper. MATERIALS

AND

METHODS

Specimen preparafion.-Hb-F was obtained from cord blood of six newborn babies and the amounts of Hb-F as detected by alkaline denaturation method of Singer et al. [lo] varied from 70-80 per cent. The abnormal Hb-E is present in heterozygous or homozygous condition in a significant portion of the population in S.E. Asia [l]. All Hb-E samples, used in these investigations, were obtained from one female subject who was homozygous in this abnormal haemoglobin (EE) and whose blood contained 98 per cent Hb-E and 2 per cent Hb-F as assayed by the electrophoretic method. Haemoglobin solutions were prepared by standard techniques, following repeated

Fig. O.--Electron fication x 200,000. Experimental

micrograph

Cell Research

of haemoglobin-E

34

molecules,

stained

negatively

with

PTA.

Magni-

Elecfron microscopy of HB-molecules

Esperimental

327

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Reseurch

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328

D. N. Misra

et al.

washing of red cells with normal saline and lysis of the cells with distilled water. After precipitation of the cell debris by centrifugation at 3000 rpm for 30 min clear Hb solution was collected from the top. Concentrations of the samples were measured from the absorption at 540 rnp by a Zeiss Spectropbotometer following the method of Donaldson et al. [5]. Ultraviolet absorption spectrum for the different samples were also obtained with the help of the same spectrophotometer. The characteristic resolved tryptophan notch of the foetal haemoglobin was found at 288.5 rnp while the abnormal haemoglobin E and the normal haemoglobin A showed an unresolved inflection at 290.5 mp (as shown in Fig. 1). Holed collodion films coated with a thin layer of carbon were used as substrate in electron microscopy. Hb solution was allowed to settle on the grids for about 10 min, the excess fluid was then drawn off by a filter paper and the grids washed with distilled water. Best results were obtained at a protein concentration of about lOA g/ml. Staining.--Staining was done exclusively with 2 per cent phosphotungstic acid, whose pH was adjusted with N/10 KOH solution. PTA was dropped on the grids, with deposited Hb molecules, and drawn off after about 5 min. Phosphotungstates with pH values 7.4, 7, 6.5, 6 and 5 were tried. Micrographs with good contrast were obtained with PTA at pH 7. Spectrophotometric studies under these conditions showed no evidence of any denaturation. IlZicroscopy.-Electron micrographs were taken by Siemens Elmiskop I at 60 kV and at an electronic magnification of 40,000. Apertures of diameters 30 p were used exclusively in these investigations.

OBSERVATIONS

AND

DISCUSSIONS

Figs. 2 and 3 represent electron micrographs of negatively stained Hb-E. while Fig. 4 is an electron micrograph from similarly prepared Hb-F. Typical molecules of haemoglobins, marked in these figures by circles, show characteristic shapes and substructures. The most striking feature of many of these molecules is the presence of a hole (e.g. particles G,. P,, F,, Ha. L,, Ma, Qa, Da, E, in Figs. 2, 3 and 4). Sometimes two, three or more particles are seen to be lying close together, when the holes of individual particles may be distinguished, i.e., the particles lie on the substrate similarly oriented (e.g. H,, L, in Fig. 3). In other instances the hole is not visible; the particles seem to be pearshaped and divided into two unequal halves by two lines radiating from a common point (e.g. A,, B,, F,, C,). Probably there are depressions along

Fig. 3.-Electron Magnification Experimental

micrograph :< 200,000. Cell Research

of

34

haemoglobin-E

molecules,

stained

negatively

with

PTA.

329

Electron microscopy of HB-molecules

Experimental

Cell

Research

34

330

D. N. Misra

Fig. I.-Electron micrograph cation x 200,000.

of haemoglobin-F

et al.

molecules, stained negatively

with PTA. Magnifi-

the lines where the a chains and p chains meet; and when the stain settles in them, they appear as faint lines in the electron micrographs. While some of the molecules (e.g. G,, F,, MS) have an approximately ellipsoidal appearance, in many instances, the shape is very asymmetrical and pointed towards one end which do not seem to agree with Perutz model. Typical amongst these are C,, D,, E,. K,, N,, O,, I,, 0,. A,, B,. F,. etc. In the case of particles C,, D,, I,, O,, A,. B,, three faint lines seem to radiate from a common point dividing them into three segments, while particles E,, J,, Mr. F,, etc. seem to be divided into four subunits by two faint lines crossing each other over the surface of the molecules. The average of the maximum and minimum dimensions of Hb-E (37) and Hb-F (26) molecules were 78 A x 62 .$ and 75 A x 65 A respectively. These dimensions are somewhat bigger than those deduced from X-ray diffraction. Valentine [ 111 reported an increase in the volume of some protein molecules when micrographed after negative staining. The explanaExperimental

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34

Electron

microscopy

of HB-molecules

331

I~~~~/~

Fig. 5.-Row 1: Negatively Hb-E molecules (400,000

stained x ); Row

Hb-F molecules (400,000 x ); Rows 2 and 3: negatively 4: shadowed Hb-A molecules (216,000 i: ).

stained

tion of this increase as given by him was that these molecules had an open structure through which phosphotungstic acid entered them and caused an expansion in volume. This explanation was corroborated by the fact that the molecules showed little contrast in the micrographs suggesting an internal uptake of PTA. But the intense staining of the haemoglobin molecules in the present case rules out this possibility. The shapes of many of the Hb molecules resemble those of individual subunits of erythrocruorin molecules reported by Levin [6]. Resides the typical molecules, some smaller particles of dimensions 2640 A are also present in the field. These may be the dissociation products of the intact molecules, their number depending on the pH value of the PT.4 and the time of staining with it. Their sizes are of the same order as those of the subunits of horse Hb molecules micrographed by Levin [7] after treatment with uranyl acetate at pH 4.3. Experimental

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332

D. N. Misra et al.

Fig. 5, rows 1, 2 and 3, shows some of the resolved fine structures of the stained molecules of Hb-E and F at a still higher magnification. Row 4 of this figure shows some Hb-A molecules micrographed after the usual shadowing with 5 A of platinum. Some of these shadowed molecules also show the typical hole and the substructures similar to those observed in the case of stained preparation. SUMMARY

Molecules of human haemoglobins E and F have been micrographed after staining with phosphotungstic acid. Many of the molecules seem to have a hole at the centre. While some of the molecules have an ellipsoidal appearance, in many instances, the shape is very asymmetrical and pointed towards one end. Many other molecules appear to be pear-shaped and divided into unequal halves by lines radiating from the centre. The average dimensions of the stained molecules appear to be about 76 x 63 A.U. Two of the authors (D. N. Misra and A. B. Sanyal) are indebted to the Ministry of Scientific Research and Cultural Affairs, Government of India, for the award of Research Training Scholarships. REFERENCES M., BIRD, G. W. G., LEHMANN, H., MOIJRANT, -4. E., THEIN, H. MASINGHE, R. L., J. Physiol. 130, 56 (1955). BRAGG, W. L. and PERUTZ, M. F., Proc. Roy. Sot. A 225, 315 (1954). CHATTERJEE, S. N., SADHUKHAN, P. and CHATTERJE.~, J. B., J. Biophys. Biochem.

1. AKSOY,

2. 3.

(1961). A. F.,

H., PEIXJTZ, 161 (1962). R., SISSON, R. B., KING, 5. DONALDSON, Larmt 1, 874 (1951). 6. LEVIN, O., J. Mol. Biol. 6, 95 (1963). ibid. 6, 158 (1963). 7. a. PERUTZ, M. F., ROSSXANS, 11. G., CULLIS, 4.

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D. N. and CHATTERJEA. Philadelphia, 2, T-i L., Blood 6, 429 (1951).

CHERNOF~, A. F. and SINGER, R. C., Nature 184, 1838 (1959).

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