Effect of environmental factors on the kinetics of insulin fibril formation: Elucidation of the molecular mechanism

In the search for the molecular mechanism of insulin fibrillation, the kine tics of insulin fibril formation were studied under different conditions us ing the fluorescent dye thioflavin T (ThT). The effect of insulin concentra tion, agitation, pH, ionic strength, anions, seeding, and addition of 1-ani linonaphthalene-8-sulfonic acid (ANS), urea, TMAO, sucrose, and ThT on the kinetics of fibrillation was investigated. The kinetics of the fibrillation process could be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elon gation). The addition of seeds eliminated the lag phase. An increase in ins ulin concentration resulted in shorter lag times and faster growth of fibri ls. Shorter lag times and faster growth of fibrils were seen at acidic pH v ersus neutral pH, whereas an increase in ionic strength resulted in shorter lag times and slower growth of fibrils. There was no clear correlation bet ween the rate of fibril elongation and ionic strength. Agitation during fib ril formation attenuated the effects of insulin concentration and ionic str ength on both lag times and fibril growth. The addition of ANS increased th e lag time and decreased the apparent growth rate for insulin fibril format ion. The ANS-induced inhibition appears to reflect the formation of amorpho us aggregates. The denaturant, urea, decreased the lag time, whereas the st abilizers, trimethylamine N-oxide dihydrate (TMAO) and sucrose, increased t he lag times. The results indicated that both nucleation and fibril growth were controlled by hydrophobic and electrostatic interactions. A kinetic mo del, involving the association of monomeric partially folded intermediates, whose concentration is stimulated by the air-water interface, leading to f ormation of the critical nucleus and thence fibrils, is proposed.