Haemoglobin molecules isolated from normal human subjects have been directly micrographed under the electron microscope following in general Hall's technique. The average height (h) and the widths along (wn) and perpendicular (wx) to the shadow direction of the molecules have been measured as 56.5 -4-6.6 A, 122.7 -4-15 A, and 120.9 ± 20 A, respectively. The exaggeration in the molecular widths due to the deposition of metal cap ranges between 60 to 70 A. The probable resolution of the substructure of the molecule, e.g., presence of "holes" and dimples, in the present electron microscopic evidence has been discussed. The electron microscopic results on the size of the individual haemoglobin molecules are in satisfactory agreement with the recent x-ray diffraction model of Perutz and his associates for horse haemoglobin.The haemoglobin molecule, the carrier of oxygen in blood, has been studied extensively by different authors. Its molecular weight is known very accurately (mol. wt. = 66,700) and the molecular structure has been determined from the x-ray diffraction studies of Bragg, Perutz, and their associates (1). According to these authors, the haemoglobin molecule is ellipsoidal in shape and has dimensions of 53 X 53 > 71 A in the hydrated condition and 45 X 45 X 65 A in the dry condition. Small angle x-ray scattering data are, however, not in very good agreement with the above model. Fournet (2) obtained a radius of gyration, R = 23 A, for horse haemoglobin. The radius of gyration corresponding to the x-ray diffraction model of dry haemoglobin molecule is 20 A and the axial ratio is 1.4. X-ray scattering data of Ritland, Kaesberg, and Beeman (3) and Rothwell (4) give an axial ratio of 1.5. The scattering curve of Rothwell (4) corresponds to an ellipsoidal molecule of dimensions 56 X 56 X 84 A. Very recently Perutz et al. (5) considered this problem again and obtained a revised model of the molecule. In this model, the molecule looks like an ellipsoid of dimensions 64 X 55 X 50 A but has a dimple on the surface in the central region and a hole passing right through the molecule. In the present work, attempts have been made to micrograph these molecules directly under the electron microscope, measure the dimensions, and find their agreement with the indirect physicochemical data.
MATERIALS AND METHODSOxalated red blood cells, collected by venipuncturc from normal human subjects, were washed with cold isotonic saline four times and then hemolysed with twice their volume of distilled water and 0.4 volume of toluene. The hemolysed cells were spun down at 1,000 g for 30 minutes and the clear haemoglobin solution was pipettcd off. For further clarification, the haemoglobin solution thus obtained was centrifuged at 10,000 g for 15 minutes.The specimens for electron microscopic studies were prepared in general according to Hall's method (6). The purified human haemoglobin solution so obtained was dissolved in a buffer containing 0.05 M ammonium carbonate and O. 1 M ammonium ace-113 on
