Chemical Composition of Human Myoglobin 



affinity of myoglobin for oxygen the above procedure leads to the 

 formation of crystals of metmyoglobin. 



In order to obtain the best crystals it is necessary to control the 

 temperature and to protect all solutions from dust and from any 

 possible colloidal impurities. We are, however, convinced that the 

 most important factor in the production of good crystals is the state of 

 the protein itself. Similar observations have been made with methaemo- 

 globin by J. Boyes-Watson, E. Davidson, and M. F. Perutz 13 . 



By these procedures crystalline samples of myoglobin from several 

 human skeletal muscles have been obtained {Figures 2, 3, and 4). It 

 should be noted that the preparation of Figure 2 was obtained from the 

 muscles of a healthy man, removed after a traumatic accident. The 

 preparation of Figure 3 was made from the muscles of a man suffering 

 from Leo-Buerger's disease, only those muscles which appeared to be 

 in good condition being taken ; in cases such as this myoglobin 

 crystallizes with difficulty, and many of our attempts were unsuccessful 

 — probably the anoxic state, the deficiency of pigment, and even the 

 denaturation of the protein, are responsible for this state of affairs. 

 The preparation of Figure 4 was obtained from the limb muscles in a 

 case of sarcoma, only muscles far removed from the site of the tumour 

 being taken ; crystallization was good although there was considerable 

 contamination from the lymphatic ganglia. 



The micrographs show that the myoglobin crystals obtained from 

 the various preparations have very much the same form. The crystals 

 of metmyoglobin are definitely birefringent and consist of very thin 

 needles which unite in more or less parallel bundles or in spheroidal 

 aggregates of radiating fibres. The birefringence is negative, a being 

 parallel to the long axis of the needle. There is straight extinction and 

 marked pleochroism, a' (brown-reddish) being parallel to the needle 

 axis and y' (pale yellow) being perpendicular to it. The refractive index 

 is greater than 1-514 when compared with balsam in xylene solution. 



In order to investigate the chemical constitution of myoglobin, 

 crystals from normal muscles were dissolved in water, the protein was 

 carefully denatured by heating, and ammonium sulphate was com- 

 pletely removed. Fat was removed with alcohol and ether, and the 

 protein was dried at 100°C and finally kept in a desiccator until its 

 weight was constant. The following analyses were then carried out : 

 nitrogen distribution by a micro-modification of the Van Slyke 

 method 2 , sulphur and iron content, and finally the content of various 

 amino-acid residues by micromethods previously described 2 ' 14 . These 

 require only 300 mg of protein in all. For comparative purposes 

 analyses were also made of microcrystalline samples of human haemo- 

 globin prepared by the methods of Drabkin 15 . 



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