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Preliminary studies on the renaturation of denatured catfish (Clarias gariepinus) glutathione transferase Yetunde Adedolapo Ojopagogo • Isaac Olusanjo Adewale • Adeyinka Afolayan

Received: 27 March 2013 / Accepted: 31 May 2013 / Published online: 9 June 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Purified juvenile catfish (Clarias gariepinus) glutathione transferase (cgGST) was denatured in vitro and renatured in the absence and presence of different concentrations of endogenous or xenobiotic model substrates. Protein transitions during unfolding and refolding were monitored by activity measurement as well as changes in protein conformation using UV difference spectra at 230 nm. Gdn-HCl at 0.22 M caused 50 % inactivation of the enzyme and at 1.1 M, the enzyme was completely unfolded. Refolding of cgGST main isozyme was not completely reversible at higher concentrations of Gdn-HCl and is dependent on protein concentration. An enzyme concentration of 30 lg/ml yielded 40 % percentage residual activity in the presence of glutathione (GSH), regardless of the concentration that was present as opposed to 30 % obtained in its absence. The xenobiotic model substrate, lindane, appears to have no effect on the refolding of the enzyme. In summary, our results show that GSH assists in the refolding of cgGST in a concentration-independent manner and may be involved in the same function in vivo whereas the xenobiotic model substrate does not.

Y. A. Ojopagogo
I. O. Adewale (&) Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria e-mail: [email protected] A. Afolayan Department of Medical Biochemistry, College of Medicine, Ekiti State University, Ado Ekiti, Nigeria

Keywords Glutathione transferase
Unfolding
Refolding
Stability
Glutathione

Introduction Glutathione transferases, GSTs (EC 2.5.1.18), are a multigene family of multifunctional proteins. This family of protein, among few other less pronounced functions, catalyzes the conjugation of reduced glutathione to a wide variety of compounds with reactive electrophilic centers, and the conjugation reaction which neutralizes these electrophilic sites and render the products more water-soluble is viewed as the archetypal functional role of the enzyme involving the detoxication of xenobiotics (Atkinson and Babbit 2009; Gupta and Ratthaur 2005). GSTs are widely distributed in nature as they are found in virtually all organisms, where they exhibit a high degree of polymorphism (Nathaniel et al. 2003). As such, they have been classified into species-independent classes namely alpha, mu, pi, theta, omega, zeta, sigma, delta, kappa, phi, tau, and beta etc. (Adewale and Ojopagogo 2010). According to their cellular localization, GSTs may be divided into cytosolic, mitochondrial, and microsomal GST. Structurally, all known cytosolic GSTs are either homodimer or heterodimer (Mannervik et al. 2005) and each subunit of GST usually contains two binding sites; the G (glutathione) and the H (hydrophobic) binding sites. The G site, as the na