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Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
Purpose: Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions.
Methods: Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism.
Results: Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs.
Conclusion: Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
Late embryogenesis abundant (LEA) proteins are related to cellular dehydration tolerance. Most LEA proteins are predicted to have no stable secondary structure in solution, i.e., to be intrinsically disordered proteins (IDPs), but they may acquire alpha-helical structure upon drying. In the model plant Arabidopsis thaliana, the LEA proteins COR15A and COR15B are highly induced upon cold treatment and are necessary for the plants to attain full freezing tolerance. Freezing leads to increased intracellular crowding due to dehydration by extracellular ice crystals. In vitro, crowding by high glycerol concentrations induced partial folding of COR15 proteins. Here, we have extended these investigations to two related proteins, LEA11 and LEA25. LEA25 is much longer than LEA11 and COR15A, but shares a conserved central sequence domain with the other two proteins. We have created two truncated versions of LEA25 (2H and 4H) to elucidate the structural and functional significance of this domain. Light scattering and CD spectroscopy showed that all five proteins were largely unstructured and monomeric in dilute solution. They folded in the presence of increasing concentrations of trifluoroethanol and glycerol. Additional folding was observed in the presence of glycerol and membranes. Fourier transform infra red spectroscopy revealed an interaction of the LEA proteins with membranes in the dry state leading to a depression in the gel to liquid-crystalline phase transition temperature. Liposome stability assays revealed a cryoprotective function of the proteins. The C- and N-terminal extensions of LEA25 were important in cryoprotection, as the central domain itself (2H, 4H) only provided a low level of protection.