Fibrillation in Human Serum Albumin Is Enhanced in the Presence of Copper(II) Nitin K. Pandey, † Sudeshna Ghosh, † and Swagata Dasgupta* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India ReceiVed: April 29, 2010; ReVised Manuscript ReceiVed: June 28, 2010 The aggregation process in proteins is governed by several factors such as temperature, pH, presence of electrolytes, denaturants, and metal ions. Here, we report the role of Cu(II) in inducing rapid fibrillation in human serum albumin. We have monitored this process via UV-vis spectroscopy, fluorescence spectroscopy, circular dichroism, -potential measurements, electron paramagnetic resonance studies, fluorescence microscopy, and field emission scanning electron microscopy. Images show a fibrillar network of human serum albumin in the presence of Cu(II) in 60% ethanol incubated at 65 °C at physiological pH. All other studies also support the enhanced fibrillation in presence of Cu(II). Introduction Proteins and peptides undergo self-aggregation leading to the formation of oligomers with different morphology in vitro under specific biophysical conditions. This pathway may in turn lead to the formation of amyloid fibrils, which appear to be a generic feature of polypeptide chains. 1–3 The fibrillar morphology associated with amyloid fibrils is linked to various neurological disorders, such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases. 4,5 Protein misfolding, a stochastic event causing these diseases, generates fibers not only in the case of disease proteins but also for disease-unrelated proteins. 6 Recent studies have reported the coordination of metal ions with amyloid fibrils where Cu(II) has been purported to be central to amyloid- (A) neurotoxicity. 7–9 Copper, an essential element, plays a critical role in human metabolism and exists in the body in either a chelated form or bound to proteins. Apart from a role in the modulation of the fibrillation pathway, Cu(II) has been shown to induce aggregation of proteins. Cu(II) is known to bind to several amyloid-forming proteins and peptides and demonstrates accelerating and/or inhibiting effects on the fibrillation process depending on the protein and biophysical conditions. 10–14 Human serum albumin (HSA), a natively R-helical protein (>60%) with 17 disulfide bridges, consists of three domains, I, II, and III, each with two subdomains (A and B). 15,16 HSA plays an important role in the transportation of fatty acids, metal ions, and other physiologically important compounds. Under normal circumstances, native HSA is reluctant to form fibers due to its stabilized helical structure. 17 The formation of HSA fibrils thus requires a necessary destabilization to generate partially folded intermediates that aggregate to form fibrils. 18 This can be accomplished by lowering the pH, increasing the temperature, and adding salts, denaturants, and metal ions. Conditions under which fibrils are formed in HSA include ethanol concentrations varying from 40 to 60% and also incubation at ambient temperature after incubation at 65 °C for 6 h. 18 Information is available on the conformational transitions in HSA in the presence of aqueous ethanol solutions. 19 It has been reported that the aggregation process does not require nucleation and incubation of HSA solutions at varying pH and ionic strength, resulting in the formation of different supramolecular assemblies of HSA amyloid-like fibers. 20,21 The pH and ionic strength have also been shown to play a role in the steps leading to HSA fibrillation with the observation that fibril formation is affected by electrostatic shielding. 22 The interaction of Cu(II) with HSA has been investigated earlier, but no reference has been made to how this may affect the fibrillation process. HSA is known to coordinate Cu(II), which plays an important role in its transportation and metabo- lism in vivo. 23 Specific binding sites in the protein have also been identified, which includes an N-terminal metal binding site and also a multimetal binding site. 24,25 HSA has been reported to show picomolar affinity to Cu(II) at its N-terminal site, with the local level of HSA being projected as an important factor in regulating the availability of Cu(II) to pathological targets. 26 Amino acid residues such as His act as potential ligands for Cu(II), and it has been shown that binding essentially occurs at a single site in HSA. 27 The complex thus formed involves the R-NH 2 terminus, NH of Ala2, and NH of His3 as also its imidazole nitrogen. Considering a protein to metal ratio of 1:1, we have investigated the effect of Cu(II) on the fibrillation process of HSA. The effect of Cu(II) on the fibrillation of HSA has been investigated under conditions that require an initial destabiliza- tion followed by incubation at ambient temperature. The destabilization was achieved by taking the protein and metal ion in a ratio of 1:1 in 60% ethanol. This was followed by incubation at 65 °C at pH 7.4 for solutions of HSA with and without Cu(II). The choice of 65 °C arises from the fact that there exists a two-state transition (T m values ∼56 °C and ∼62 °C) that occurs during the denaturation of HSA corre- sponding to the sequential unfolding of the domains as mentioned in ref 20, where a T m value of 66 °C has also been reported. 28,29 This temperature is chosen to ensure partial unfolding of HSA that facilitates aggregation. HSA fibrils were obtained in the solutions with and without Cu(II) when heated at 65 °C in the presence of ethanol at pH 7.4 followed by incubation at room temperature. The solutions were monitored over time by Congo Red (CR)-based UV studies, thioflavin T (ThT) fluorescence studies that are characteristic of fibril formation in addition to circular dichroism (CD) experiments, -potential measurements, and electron paramagnetic resonance (EPR) studies. Images of the fibers were obtained through fluorescence microscopy and field emission scanning electron * To whom correspondence should be addressed. Tel: +91 3222 283306. Fax: +91 3222 255303. E-mail: swagata@chem.iitkgp.ernet.in. † Both authors contributed equally to the work. J. Phys. Chem. B 2010, 114, 10228–10233 10228 10.1021/jp103876p  2010 American Chemical Society Published on Web 07/20/2010