title: Preformed Seeds Modulate Native Insulin Aggregation Kinetics.
abstract: Insulin aggregates under storage conditions via disulfide interchange reaction. It is also known to form aggregates at the site of repeated injections in diabetes patients, leading to injection amyloidosis. This has fueled research in pharmaceutical and biotechnology industry as well as in academia to understand factors that modulate insulin stability and aggregation. The main aim of this study is to understand the factors that modulate aggregation propensity of insulin under conditions close to physiological and measure effect of "seeds" on aggregation kinetics. We explored the aggregation kinetics of insulin at pH 7.2 and 37 °C in the presence of disulfide-reducing agent dithiothreitol (DTT), using spectroscopy (UV-visible, fluorescence, and Fourier transform infrared spectroscopy) and microscopy (scanning electron microscopy, atomic force microscopy) techniques. We prepared insulin "seeds" by incubating disulfide-reduced insulin at pH 7.2 and 37 °C for varying lengths of time (10 min to 12 h). These seeds were added to the native protein and nucleation-dependent aggregation kinetics was measured. Aggregation kinetics was fastest in the presence of 10 min seeds suggesting they were nascent. Interestingly, intermediate seeds (30 min to 4 h incubation) resulted in formation of transient fibrils in 4 h that converted to amorphous aggregates upon longer incubation of 24 h. Overall, the results show that insulin under disulfide reducing conditions at pH and temperature close to physiological favors amorphous aggregate formation and seed "maturity" plays an important role in nucleation dependent aggregation kinetics.
authors.affiliation: Department of Chemistry, Michigan Technological University , Houghton, Michigan 49931, United States.
authors.forename: Colina
authors.initials: C
authors.lastname: Dutta
authors.affiliation: Department of Chemistry, Michigan Technological University , Houghton, Michigan 49931, United States.
authors.forename: Mu
authors.initials: M
authors.lastname: Yang
authors.affiliation: Department of Mechanical Engineering, Michigan Technological University , Houghton, Michigan 49931, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Long
authors.affiliation: Department of Mechanical Engineering, Michigan Technological University , Houghton, Michigan 49931, United States.|Department of Mechanical and Industrial Engineering, University of Illinois at Chicago , Chicago, Illinois 60607, United States.
authors.forename: Reza
authors.initials: R
authors.lastname: Shahbazian-Yassar
authors.affiliation: Department of Chemistry, Michigan Technological University , Houghton, Michigan 49931, United States.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Tiwari
mesh_terms: D007328:Insulin / Q000737:chemistry*; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D008958:Models, Molecular; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D007328:Insulin