Amyloid Fibril Formation and Other Aggregate Species Formed by Human Serum Albumin Association Pablo Taboada,* Silvia Barbosa, Emilio Castro, and Vı ´ctor Mosquera Laboratorio de Fı ´sica de Coloides y Polı ´meros, Grupo de Sistemas Complejos, Departamento de Fı ´sica de la Materia Condensada, Facultad de Fı ´sica, UniVersidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain ReceiVed: July 29, 2006; In Final Form: August 29, 2006 Under in vitro solution conditions where the native state is destabilized, many proteins present an abnormal structure and metabolism associated with a strong tendency to self-aggregation into a polymeric amyloid fibril structure, suggesting that this ability is a generic feature of the polypeptide chains. Such structures play a key role in different pathogenesis of neurodegenerative diseases such as Alzheimer, Parkinson, or Creutzfeldt- Jakob. Here, we report the formation of amyloid fibrils in the plasma protein human serum albumin under different in vitro conditions monitored using a combination of spectrophotometric and microscopic tecnhiques. Amyloid fibril formation, therefore, is also allowed in a protein with a high degree of structural complexity. We also infer from experimental data the existence of other protein aggregated species than fibrils, some of which seem to be formed by a structural rearrangement of the proper fibrils. Under in vitro solution conditions where the native state is destabilized, many proteins present an abnormal structure associated with a strong tendency to self-aggregation into a polymeric amyloid fibril structure, suggesting that this ability is a generic feature of the polypeptide chains. 1,2 These amyloid fibrils are polypeptide aggregates with a core formed by -strands extend transversely to the main fibril axis. 1,3 Such structures play a key role in different pathogenesis of neuro- degenerative diseases 4,5 such as Alzheimer, Parkinson, or Creutzfeldt-Jakob and, recently elucidated, in other nonpatho- genic phenomena as the transfer of genetic information or synaptic changes associated with memory. 6,7 Here, we report the formation of amyloid fibrils in human serum albumin (HSA) under different in vitro conditions monitored using a combina- tion of spectrophotometric and microscopic techniques. Amyloid fibril formation, therefore, is also allowed in a protein with a high degree of structural complexity. 1,8 We also infer from experimental data the existence of other protein aggregated species than fibrils, some of which seem to be formed by a structural rearrangement of the proper fibrils. Thus, the observa- tions lead to the suggestion that the aggregated form may represent a generic form of the protein, and point out the necessity of a complete knowledge of these self-association phenomena in order to design strategies to prevent pathologycal amyloidogenic conditions. HSA is an all-R-protein which constitutes approximately half of the total blood protein, acting as a carrier from bloodstream to tissues, but also maintains the osmotic pressure and plays a key role in coagulation and thrombosis. It consists of 585 amino acids in a single polypeptide chain with a molar mass of 66 411 g mol -1 (see Supporting Information for further details). Thus, native HSA lacks any properties suggesting a predisposition to form amyloid fibrils, since most of its sequence (>60%) is arranged in an R-helix structure, with the subsequent tightening of its structure through intramolecular interactions such as hydrogen bonds. In a screening process, pH, temperature, and ethanol con- centration as a cosolvent were varied in order to determine the type of aggregates formed under different conditions, with special emphasis on those where partial destabilization of the nativelike structure of HSA occurred, which are thought to be a crucial factor in the amyloid formation by globular proteins. 1,2 The formation of these protein aggregates would suggest that, under proper solution conditions, inter- and intramolecular interactions are conveniently modified involving that the ther- modynamic stable state would be the aggregated one, whereas the supposed correct folding of HSA would not signify more than a metastable state, as suggested by Ganzit in a pioneering paper. 9 Fibril formation was found (a) in the presence of ethanol concentrations between 40 and 60% (v/v) at protein concentra- tions larger than 1% (w/v) at room temperature, (b) after incubation at 65 °C for 6 h, and (c) at pH 2 and 7 after incubation at 65 °C for 6 h, followed by 7- or 30-day incubation at ambient temperature (Figure 1a). The kinetics of fibril formation was not analyzed in detail. Other kinds of aggregates were found under other solution conditions (see below). Previous studies had shown the formation of fibril structures in bovine serum albumin (BSA) after incubation at high temperatures, although the compositions of these aggregates were not ana- lyzed. 10,11 However, the formation of amyloid fibrils in the presence of ethanol is less known. 12 Ethanol increases the R-helix content of HSA up to concentrations of 20% (v/v), enhancing intramolecular interactions. However, at higher * To whom correspondence should be addressed. E-mail: fmpablo@usc.es. Phone: 0034981563100, Ext. 14042. Fax: 0034981520676. 20733 2006, 110, 20733-20736 Published on Web 09/27/2006 10.1021/jp064861r CCC: $33.50 © 2006 American Chemical Society