Published: February 24, 2011 r2011 American Chemical Society 2617 dx.doi.org/10.1021/jp110367t | J. Phys. Chem. B 2011, 115, 2617–2626 ARTICLE pubs.acs.org/JPCB Detecting the Early Onset of Shear-Induced Fibril Formation of Insulin in situ Grant T. Webster,* ,† Jonathan Dusting, ‡ Stavroula Balabani, ‡ and Ewan W. Blanch † † Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. ‡ Experimental and Computational Laboratory for the Analysis of Turbulence (ECLAT), School of Natural and Mathematical Sciences, King’s College London, Strand, London WC2R 2LS, U.K. ’ INTRODUCTION The misfolding and aggregation of proteins can lead to amyloidosis, where proteins can transform into insoluble amy- loid fibrils or plaques that deposit in a variety of organs and tissues. 1-3 Amyloidosis has been linked to the onset of neuro- degenerative diseases and other human disorders such as Alzhei- mer’s and Parkinson’s diseases, the spongiform encephalopathies such as Creutzfeldt-Jakob disease, and Type II diabetes. 4,5 Although the various proteins involved in these diseases have different characteristic folds which are native to the proteins, the structures of the fibrils formed are similar, being mostly β-sheet, 6 where the peptide backbone is stabilized by intermolecular bonds. 7,8 Protein aggregation is also of concern in the biotech- nology industry, as it often leads to product fouling. For these reasons, understanding the process of fibrillogenesis and resultant fibril morphology are currently the subject of considerable research interest. 9 A number of probing methods have been employed to induce fibrillization, including changes in pH or heating. While the effects of pH and temperature on protein misfolding and fibril formation are well established, 10-12 the manner in which mechanical strain properties affect fibril formation has not been thoroughly characterized, despite the importance of mechanical stresses, including fluid stresses, in physiological and processing environments. A number of proteins have been shown to exhibit altered fibril formation characteristics due to the presence of flow, including β- lactoglobulin, 13,14 insulin, 15,16 amyloid-β 1-40 , 17-19 whey protein, 20,21 and hen lysozyme. 22 Previous studies by Hill et al., 23 among several others, have shown that flow promotes amyloid fibril formation. They discovered from in situ real time fluores- cence measurements that exposure of β-lactoglobulin to con- trolled shear flow in vitro promotes fibrillogenesis by generating precursors that act as seedlike initiators. 23 Recent real time monitoring studies of protein dynamics under controlled shear by Ashton and co-workers 24,25 demonstrated that flow can indeed induce reversible structural changes to the protein back- bone, which supports the mechanism proposed by Hill et al. 23 Other studies have shown that stirring, shaking, or mechanical agitation accelerates the rate of fibril formation 26-28 and influ- ences fibril morphology, via exposure to shear stresses and/or the promotion of surface interactions. 19,26-29 Bovine insulin is a small globular protein consisting of 51 amino acid residues and is a model for human insulin. It has a known susceptibility to structural change and aggregation under Received: October 29, 2010 Revised: January 19, 2011 ABSTRACT: A new approach is presented for detecting the early onset of amyloid fibril formation of insulin in a fluidic environment. The fibrillogenesis of insulin in a well-character- ized Taylor-Couette flow cell was analyzed in situ using Raman spectroscopy in combination with principal components anal- ysis (PCA). Raman spectra recorded using a 532.5 nm excita- tion laser revealed a more rapid fibrillogenesis process during the first 90 min of shearing than previously reported for samples exposed to flow. Bands corresponding to intermolecular H-bonded β-sheet structure of insulin at 1678, 1630, and 1625 cm -1 observed in the Raman difference spectra between unsheared insulin and sheared insulin show an increase in intensity as a function of shear exposure time, which is characteristic of fibril formation, with the first changes detected after 10 min. Additional analysis of samples removed from the flow cell after specific time periods provided conformation of the flow-enhanced fibrillogenesis process, including the detection of early fibril formation after only 1 min of shearing. FT-IR spectra of the insulin solutions showed evolution of bands at 1673 and 1633 cm -1 from an increase in H-bonded β-turn and β-sheet structures, respectively, while fluorescence emission spectra detected the presence of a new emission band at 482 nm. TEM images confirmed the early onset of fibril formation at 1 min shear exposure, before a maturation and concentration increase of fibrils with further shearing. This study highlights the ability of fluid flows to accelerate insulin fibril formation, which has important implications for biotechnology applications such as the purification process of insulin therapeutic drugs in the pharmaceutical industry, as well as the use of optical-based methods for detecting fibrillogenesis.