Jack bean urease (EC 3.5.1.5) aggregation monitored by dynamic and static light scattering Cristian Follmer a , Fabiano V. Pereira b , Na ´dya P. da Silveira b , Ce ´lia R. Carlini a, * a Department of Biophysics, IB, and Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonalves 9500, Porto Alegre CEP 91501970, RS, Brazil b Institute of Chemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre CEP 91501970, RS, Brazil Received 9 January 2004; received in revised form 24 March 2004; accepted 25 March 2004 Available online 18 May 2004 Abstract Aggregation of jack bean urease (JBU) is involved in many alterations of its biological properties, notably the ureolytic and entomotoxic activities. In order to investigate this phenomenon, protein aggregates were characterized by dynamic (DLS) and static light scattering (SLS) spectroscopies through determination of apparent hydrodynamic radii, the average molecular masses, radii of gyration and second virial coefficients. No effect of disulfide reducing agents on protein association was observed contrasting with previous reports implicating their function in the prevention of JBU aggregation. The influence of freeze – thawing cycles on protein aggregation was also investigated. Our results showed that after freeze – thawing cycles the native form of JBU with apparent hydrodynamic radius of 7 nm and radius of gyration of 12 nm is replaced by high-order oligomers and this aggregation is not reverted neither by dithiothreitol (DTT) treatment nor by high concentration of salts. Altogether the data help to understand the complex behavior of JBU in solution and may correlate with the diversity of biological properties of this enzyme. D 2004 Elsevier B.V. All rights reserved. Keywords: Urease; Oligomerization; Freeze – thawing; Hydrodynamic radius; Radius of gyration 1. Introduction Urease (EC 3.5.1.5; urea amidohydrolase) is a nickel dependent enzyme [1] that catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. In 1926, jack bean urease (JBU) was crystallized by Sumner [2] and these first crystals of a characterized enzyme demonstrated the pro- teinaceous nature of the enzymes. JBU exists as monomers, trimers and hexamers of identical 91 kDa chains, each containing two nickel ions per subunit [3]. Although JBU was the first protein ever crystallized, its structure has yet to be determined. Heterogeneity, insolubility, molecular flexi- bility or polydispersity of JBU in solution could be related to difficulties in obtaining crystals suitable for X-rays diffraction studies [4]. Many works have investigated the oligomerization/ag- gregation behavior of JBU and how this relates to its catalytic properties [5–7]. It has been shown that different jack bean seeds contain distinct molecular forms of JBU and the multiplicity of urease isoenzymes, in conjunction with their interconvertibility, may underlie many catalytic com- plexities of this enzyme [8]. Recent studies of our group have shown that JBU dis- plays other biological activities, which are unrelated to its ureolytic property, such as activation of blood platelets, interaction with glycoconjugates and entomotoxic activity, this latter suggesting that urease(s) may be involved in plant defense [9–11]. Interestingly, it was observed that many of these biological activities are modified under storage con- ditions in which protein oligomerization takes place (un- published). Moreover, aggregation of JBU is believed to impact the shelf life of diagnostic kits based on this enzyme [12]. Aggregation is also a major obstacle to useable crystal growth [13] and may be the reason of the difficulties encountered for growing crystals of JBU capable of diffract- ing X-rays at low angles [14,15]. 0301-4622/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2004.03.009 * Corresponding author. Tel.: +55-51-3316-7606; fax: +55-51-3316- 7003. E-mail address: ccarlini@ufrgs.br (C.R. Carlini). www.elsevier.com/locate/bpc Biophysical Chemistry 111 (2004) 79– 87