M. F. PERUTZ 



properties, which enabled me to calculate the electron density distri- 

 bution in projection along a line through the molecule. Finally, the 

 structure happens to be one of those exceptional types mentioned in 

 the article with J. C. Kendrew (p. 149), where the vector structure 

 bears a strong resemblance to the real one. It was the parallelism of 

 the polypeptide chains in each haemoglobin molecule, and of all the 

 molecules in the crystal, which facilitated the interpretation of the 

 three-dimensional Patterson synthesis. This synthesis forms the main 

 theme of the present article, but before passing on to it I shall review 

 briefly some of the more important properties of the haemoglobin 

 crystals, and describe their molecular structure as we saw it before the 

 results of the Patterson synthesis became available. Both this work 

 and the results of the Patterson synthesis have been published in full 

 elsewhere (J. Boyes- Watson, E. Davidson and M. F. Perutz 1 ; M. F. 

 Perutz 2 ; henceforth referred to as J and //). For readers who are 

 unfamiliar with the methods of x-ray crystallography and especially 

 with the meaning of Patterson syntheses, a brief outline of x-ray 

 analysis as applied to the study of crystalline macromolecules is 

 provided in this volume (Kendrew and Perutz, this vol. p. 161). 



Over 50 per cent of the volume of wet haemoglobin crystals consist 

 of liquid of crystallization. More than a third of this is water ' bound ' 

 to the protein molecules and therefore not available as solvent to 

 electrolytes, but the remainder is free and acts as a medium through 

 which a variety of ions can diffuse without damaging the structure 

 of the crystals 3 . This sponge-like character is a unique and peculiar 

 property of protein crystals, and can be used with great advantage in 

 their x-ray analysis. For instance, by allowing heavy ions to diffuse 

 into the liquid of crystallization, the x-ray scattering power of the 

 liquid can be enhanced relative to that of the protein ; in effect, the 

 liquid regions in the crystal can be ' stained ' as far as their x-ray 

 scattering power is concerned, with resulting changes in the relative 

 intensities of certain diffracted rays. These changes can give important 

 information about the distribution of liquid in the unit cell, and hence 

 about the shape of the molecules. 



Protein crystals suspended in solutions of different electrolytes or in 

 atmospheres of different humidity can be made to swell or shrink. 

 A great deal was learnt by comparing the diffraction patterns of the 

 same crystal at different states of swelling and shrinkage. It was 

 found, for instance, that the haemoglobin molecules themselves were 

 rigid and impenetrable to liquid, and that it was merely their relative 

 arrangement and the distances between them which alter during 

 swelling and shrinkage. It was also through shrinkage experiments 

 that the layer structure of the crystals was first discovered. 



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