Static and dynamic light scattering were used to investigate the effects of L-arginine, commonly used to inhibit protein aggregation, on the initial aggregation kinetics of solutions of bovine insulin in 20% acetic acid and 0.1？M NaCl as a model system for amyloidosis. Measurements were made as a function of insulin concentration (0.5–2.0？mM), quench temperature (60–85°C), and arginine concentration (10–500？mM). Aggregation kinetics under all conditions had a lag phase, whose duration decreased with increasing temperature and with increasing insulin concentration but which increased by up to a factor of 8 with increasing added arginine. Further, the initial growth rate after the lag phase also slowed by up to a factor of about 20 in the presence of increasing concentrations of arginine. From the temperature dependence of the lag phase duration, we find that the nucleation activation energy doubles from 1 7 ± 5 to 3 6 ± 3 ？kcal/mol in the presence of 500？mM arginine. 1. Introduction Conformational misfolding under destabilizing conditions and subsequent beta-sheet amyloid fibril formation has been shown to be a very common, perhaps generic, property of proteins [1, 2]. In a number of proteins, such aggregation is pathological leading to over 40 different neurodegenerative or systemic diseases [3, 4] such as Alzheimer’s, Parkinson’s, Huntington’s, and type II diabetes. It is becoming more apparent that the toxic species of many proteins is formed in the early stages of soluble aggregation. Such proteins include Aβ, for which an annular protofibril form has recently been isolated [5], and insulin [6, 7]. Much recent effort has been devoted to understanding the details of aggregate formation and also to find ways to control or inhibit this process. In the case of insulin, the motivation for such studies includes improving its pharmacological use in treating diabetes, as well as using it as a model system for studying amyloid aggregation. Insulin, a key component in glucose metabolism, is a 51-residue protein consisting of two chains linked by disulfide bonds. Since the pioneering studies of David Waugh in the 1940’s and 50’s [8, 9], it has been known that, at elevated temperatures at low pH, insulin aggregates to form fibers that precipitate and/or form gels following a nucleation and elongation reaction. Despite this early finding and many subsequent studies [10–23], the molecular mechanism of insulin aggregation is not fully understood. Aggregation of insulin has been studied under a variety of solvent conditions and with added cosolutes. Added ethanol
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