With the increase in the time of incubation, the negative ellipticity values at 222 nm and 208 nm also decrease, which indicates increased order in the AB1010 VEGFR/PDGFR inhibitor secondary structure of insulin. In order to have a quantitative analysis of the change in secondary structure of insulin during fibrillation process, we have de-convoluted the far UV-CD data with the help of CDNN software. After 100 min of incubation, the helix content of insulin decreased to 39% with little increase in b-sheet structure. Whereas, the secondary structure of insulin in presence of NK9, remained almost unchanged for 180 min of incubation. At 240 min of incubation, helix content of insulin in presence of NK9 decreased only to 42%. This implies that NK9 helps insulin to retain its secondary structure for a prolonged period of time. Appearance of insoluble aggregates makes it difficult to continue the CD measurement beyond 100 min of incubation for insulin alone and 240 min of incubation, for insulin in presence of NK9. Nevertheless, NK9 does not have a well-defined structure even in the presence of insulin. The association states of insulin in the presence and absence of NK9 were determined by the size exclusion chromatography using TSKgel SuperSW2000 HPLC column. The column was precalibrated using size exclusion marker proteins b-amylase, ADH, BSA, carbonic anhydrase, lysozyme, and ribonuclease. The calibration data fitted nicely into a linear equation. Since incubation of insulin samples for fibrillation experiment were done using citrate phosphate buffer of pH 2.6, the HPLC column was equilibrated with this buffer for experiment with insulin. It was confirmed by using BSA and CHIR-99021 lysozyme that both proteins retained their globular shape as retention time at this acidic pH did not significantly change from those at pH 7.0. Aliquots of insulin solution at different time points of incubation were centrifuged at 40006g force to remove visible turbidity. Supernatant was loaded on to the HPLC column. Prior incubation of insulin in citrate phosphate buffer showed a retention time of 16.9 ml. This retention time corresponds to the trimeric structure of insulin that corroborates well with earlier findings by Banga and co-workers. Retention time of insulin remained unchanged for 1.5 hr with concomitant decrease in its absorbance value. The increase in incubation time up to 2 hr, shifted the retention time of insulin to 17.9 min that corresponds to the monomeric insulin. After 2 hr of incubation, fluorescence emission intensity just started to increase implying that active nucleus of insulin fibrils was formed just after 2 hr of incubation.