Effect of temperature and pH on the secondary structure and processes of oligomerization of 19 kDa alpha-zein Vanessa Cabra, Roberto Arreguin, Rafael Vazquez-Duhalt, Amelia Farres ⁎ Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, 04510 México D. F., México Instituto de Química, Universidad Nacional Autónoma de México, 04510 México D. F., México Instituto de Biotecnología, Universidad Nacional Autónoma de México, 04510 México D. F., México Received 9 February 2006; received in revised form 29 March 2006; accepted 3 April 2006 Available online 7 April 2006 Abstract Highly hydrophobic protein Z19 zein shows a tendency towards oligomerization. The role of temperature and pH on the oligomerization process was studied monitoring the secondary structure content and the appearance of aggregates by Circular Dichroism Spectroscopy (CD) and Dinamic Light Scattering (DLS). Z19 zein suffers irreversible thermal denaturalization, as demonstrated by far-UV CD measurements. DLS data indicate that this denaturalization is accompanied by oligomerization processes which are strongly dependent on temperature. The aggregates that appear when the sample is heated maintain a certain amount of their native structure. Oligomers, showing high stability to temperature changes and other denaturing conditions with molecular weights of 45, 66 kDa and higher, were detected by SDS-PAGE. The secondary structure strongly depends on pH. Thus, at pH above pI (6.8), all the protein structure is in alpha helix. The formation of disulfide bonds plays an important role in the aggregation process, since most of the sulfhydryls in the protein (97.52%) form disulfide bonds and only 2.47% of them are free and superficially exposed. The sensitivity towards thermal denaturalization is also affected by pH rises. © 2006 Elsevier B.V. All rights reserved. Keywords: Alpha-zeins; Protein aggregation; Circular Dichroism; Dinamic light scattering; Disulfide bond 1. Introduction The factors that affect the secondary and tertiary structures of highly hydrophobic proteins, such as prolamins, are far less understood than those that affect water-soluble proteins [1,2]. Zeins, corn (Zea mayz L.) prolamins, are synthesized during the endosperm development and stored in proteic bodies [3]. Zeins are classified according to their solubility, molecular weight and immunological response into α, β, γ, and δ [4–6]. α-Zeins are the most abundant (75– 85% of the total) and are divided in two types: Z19 and Z22 with approximate weights of 22 and 25 kDa respectively [7]. Several analytical efforts have been made to elucidate the molecular structure of zeins [8]. Physicochemical and structural characterization studies of the α-zeins mixture [9–15] suggest that these proteins have a high content of alpha-helix (40–60%). Nevertheless, there are no three- dimensional structures of α-zeins available. Proposed models from these studies suggest that the zeins show a compacted form within protein globules, with elongated molecular structures of prolates consisting of ellipsoids and a series of repeated and packed alpha-helixes [8]. In solution, zeins became an extended structure. It is well known that these proteins are organized in oligomers which are resistant to high temperatures and treatment with reducing reagents, and that they tend to form aggregates of high molecular weight (HMW) [16]. The aggregation process is complex, and it has not been completely elucidated, because it depends on a great number of physical and chemical parameters Biochimica et Biophysica Acta 1764 (2006) 1110 – 1118 http://www.elsevier.com/locate/bba Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD, circular dichroism; BME, beta mercapthoethanol; HMW, High Molecular Weight; DLS, Dinamic Light Scattering; EDTA, Ethilendiami- notethracetic acid; DTNB, 5′5′-dithiobis(2-nitrobenzoic acid) ⁎ Corresponding author. Tel.: +52 55 5622 5348; fax: +52 55 5622 5329. E-mail address: farres@servidor.unam.mx (A. Farres). 1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.04.002