9] X-RAY ANALYSIS OF HAEMOGLOBIN 139 



to exhibit any features which could be identified with either polypeptide 

 chains or haem groups. 



Clearly it is still the great thickness of matter projected on one plane 

 which defeats us, and it looks as though only a three-dimensional analysis, 

 giving the electron density distribution along a series of parallel sections 

 through the molecule, will finally reveal its structure. This will have to be 

 the next step. 



Dr Kendrew has already explained to you that this involves determina- 

 tion of the phase angles of each one of many thousands of X-ray reflexions, 

 which is far more diflScult than the determination of signs in the projection 

 along the two-fold axis to which our work had been confined so far. It 

 seemed especially doubtful if the phase angles could be measured with suffi- 

 cient accuracy in a molecule as big as haemoglobin, where the number of 

 sites combining with heavy atoms per unit molecular weight of protein is 

 only half as great as in myoglobin. The projection along the a axis, which 

 lacks a centre of symmetry, was therefore chosen as a test case for a later 

 three-dimensional analysis. Its successful solution by D. M. Blow is most 

 encouraging, though the accuracy of the phase angles so far obtained could 

 be much improved if one or two more sites combining with heavy atoms 

 could be discovered. 



In conclusion I should like to mention one interesting piece of informa- 

 tion about the structure of the haemoglobin molecule obtained by D. J. E. 

 Ingram, J. F. Gibson and myself.^ We determined the orientation of the four 

 haem groups in the haemoglobin molecule by electron spin resonance, using 

 a single crystal of horse haemoglobin. We found the haem groups to be 

 arranged in two pairs, related by the dyad axis of symmetry. The normals 

 to one pair of haem groups lie in the crystallographic a, b plane at an angle 

 of 32° on either side of the a axis. The normals to the other pair have a 

 similar orientation, except that they are tilted by 13° above and below the 

 a, b plane. Knowing the orientation of the haem groups tells us nothing as 

 yet about their positions, but it will make it much easier to find them by 

 X-ray analysis later on. 



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