342 J0RGEN SCHLICHTKRULL [18 



This value, 2 atoms/unit-cell, is also significant in quite other investiga- 

 tions, viz. in the ultracentrifugal studies of Cunningham, Fischer and Vestling 

 on insulin solutions.^ These authors found that the sedimentation constant 

 was independent of the zinc content when the zinc concentration varied from 

 0'2 to about 0-6-0 -8 moles of zinc per 12,000 grams of insulin. However, 

 a rise in the zinc concentration above the latter value brought about a linear 

 rise in the sedimentation constant. The minimum amount sufficient for 

 crystallization, 2 atoms of Zn/unit-cell, is equivalent to 0-7 moles of zinc 

 per 12,000 grams of insulin and it is therefore likely that the sudden rise in 

 the sedimentation constant at this value corresponds to the fact that the 

 condition for crystallization is reached at this Zn content. 



The shape of the insulin crystals depends on the species.' Recrystallized 

 pig-insulin crystals are perfectly shaped single rhombohedrons, but beef 

 insulin crystals are twin-like bodies of a very different, star-Hke appearance. 

 (It should be noted that the internal structure is rhombohedral in all cases 

 considered here.) If beef-insulin crystals are recrystallized in one of the 

 usual buffers and sodium chloride is added to the buffer, it can be observed 

 that the shape of the crystals formed depends on the concentration of sodium 

 chloride. If the concentration of sodium chloride exceeds approx. 6%, the 

 crystals attain the perfect rhombohedral shape characteristic of pig-insulin. 

 This rather abrupt change in shape is associated with a change in the mini- 

 mum amount of zinc ions sufficient for crystallization. If the buffer contains 

 so much sodium chloride, e.g. 7%, that the shape of beef insulin crystals 

 becomes rhombohedral, the minimum adequate zinc concentration — and 

 this applies also to pig-insulin — is no longer 2 but 4 atoms/unit-cell. Other 

 experiments have shown that the chloride ion is responsible for these trans- 

 formations and that the other halogen ions, with the exception of fluoride, 

 have the same effect. Nitrate, sulphate and phosphate proved to have no 

 effect. The transformations are not contingent upon the sodium ions since 

 the latter can be replaced by potassium, ammonium and calcium. 



Microscopical, goniometric measurements on a large (1-3 mm.) perfect 

 insulin rhombohedron' gave a value (114° 22' ±4' (S.E.)) which was in agree- 

 ment with the X-ray value^ (1 14° 16') for the unit-cell in wet insulin crystals. 



The kinetics of insulin crystalhzation have been studied in detail. It was 

 found that the star-shaped or twin-like beef-insulin crystals have a tendency 

 to form monodisperse suspensions. It means that the nuclei are formed 

 almost exclusively in the initial stage of crystallization where the insulin 

 concentration is greatest. The sharp and perfect pig-insulin rhombohedrons 

 and the identically shaped beef-insulin crystals formed in sahne are very 

 polydisperse. The rate of nucleation was deduced from the very peculiar 

 observation that the ratios between the total volume, total surface, total 

 length and number of the crystals remain constant in the course of crystalliza- 

 tion from the moment when the first few crystals had formed. It is possible 

 to account for the nucleation in the most simple way if — and apparently 



