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Monitoring Insulin Aggregation via Capillary Electrophoresis
Ralph E. Martin Department of Chemical Engineering, 3202 Bell Engineering Center, University of Arkansas, Fayetteville, AR 72701, USA
Department of Chemical Engineering, 2C02 Swearingen Engineering Center, University of South Carolina, Columbia, SC 29208, USA
Current address: National Institute of Standards and Technology, 100 Bureau Drive, Stop 8311, Gaithersburg, MD 20899, USA; E-Mail: email@example.com.
* Author to whom correspondence should be addressed.
Received: 22 October 2011; in revised form: 6 December 2011 / Accepted: 12 December 2011 / Published: 14 December 2011
Abstract: Early stages of insulin aggregation, which involve the transient formation of oligomeric aggregates, are an important aspect in the progression of Type II diabetes and in the quality control of pharmaceutical insulin production. This study is the first to utilize capillary electrophoresis (CE) with ultraviolet (UV) detection to monitor insulin oligomer formation at pH 8.0 and physiological ionic strength. The lag time to formation of the first detected species in the aggregation process was evaluated by UV-CE and thioflavin T (ThT) binding for salt concentrations from 100 mM to 250 mM. UV-CE had a significantly shorter (5–8 h) lag time than ThT binding (15–19 h). In addition, the lag time to detection of the first aggregated species via UV-CE was unaffected by salt concentration, while a trend toward an increased lag time with increased salt concentration was observed with ThT binding. This result indicates that solution ionic strength impacts early stages of aggregation and β-sheet aggregate formation differently. To observe whether CE may be applied for the analysis of biological samples containing low insulin concentrations, the limit of detection using UV and laser induced fluorescence (LIF) detection modes was determined. The limit of detection using LIF-CE, 48.4 pM, was lower than the physiological insulin concentration, verifying the utility of this technique for monitoring biological samples. LIF-CE was subsequently used to analyze the time course for fluorescein isothiocyanate (FITC)-labeled insulin oligomer formation. This study is the first to report that the FITC label prevented incorporation of insulin into oligomers, cautioning against the use of this fluorescent label as a tag for following early stages of insulin aggregation.
Keywords: capillary electrophoresis; ultraviolet absorbance; laser induced fluorescence; thioflavin T; insulin; oligomer; amyloid
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Pryor, E.; Kotarek, J.A.; Moss, M.A.; Hestekin, C.N. Monitoring Insulin Aggregation via Capillary Electrophoresis. Int. J. Mol. Sci. 2011, 12, 9369-9388.
Pryor E, Kotarek JA, Moss MA, Hestekin CN. Monitoring Insulin Aggregation via Capillary Electrophoresis. International Journal of Molecular Sciences. 2011; 12(12):9369-9388.
Pryor, Elizabeth; Kotarek, Joseph A.; Moss, Melissa A.; Hestekin, Christa N. 2011. "Monitoring Insulin Aggregation via Capillary Electrophoresis." Int. J. Mol. Sci. 12, no. 12: 9369-9388.