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Insulin crystals: zinc atoms in the unit-cell, 

 nucleation, growth and shape 



J0RGEN SCHLICHTKRULL 



Novo Terapeutisk Laboratorium, Copenhagen, Denmark 



The number of metal atoms (Zn, Cd, Co or Ni) present in the unit-cell 

 (M^ca. 35,000) of rhombohedral insulin crystals is an important detail in 

 the structural concept. The various values reported for metal contents in 

 the literature are quite confusing in contrast to the original ngure,^-^-^ of 

 3 atoms/cell, which is also the number of dimer elements in the cell. Since 

 the metal content of insulin crystals is actually subject to gross variations* 

 depending upon many conditions, attempts have been made to determine 

 the number of zinc atoms/unit cell which is most relevant in structural 

 considerations. 



A quantitative examination showed some interesting features of the role 

 played by zinc or the other metal ions in the crystallization. Insulin was 

 crystallized with zinc in a phosphate buffer and recrystallized in ammonium 

 acetate. The crystals contained 3 atoms of Zn/unit-cell (0-51% Zn), in per- 

 fect agreement with Scott and Fisher's experimental results. ^-^ However, 

 when the crystals were recrystallized once more, the zinc content dropped 

 to 2 atoms/cell and remained constant at that value through several sub- 

 sequent recrystallizations in ammonium acetate. If insulin is recrystallized 

 from a citrate buffer containing zinc ions — which are strongly complexed to 

 the citrate — then the crystals contain 2 atoms of zinc/unit-cell. ^ When such 

 crystals are recrystallized from a citrate buffer without the addition of zinc 

 ions, the new crystals are again found to contain 2 atoms/unit-cell, but in 

 the course of several such recrystallizations the content decreases a little 

 and at the same time the crystals deteriorate in shape and amorphous par- 

 ticles appear in the precipitates. This slight decrease in the zinc content is 

 not observed with recrystallizations from ammonium acetate and can pre- 

 sumably be accounted for by competition between insulin and citrate with 

 respect to the binding of zinc ions. It is noteworthy that the zinc content of 

 the citrate-recrystallized insulin (2 atoms/unit-cell) is not dependent on the 

 pH in the interval over which crystallization takes place (5*5<pH<7). It 

 is concluded from these experiments that among the many combinations 

 between zinc and insulin crystals, the combination containing 2 atoms/unit- 

 cell is a very strong complex and represents the minimum content of zinc 

 ions sufficient for crystallization. 



