Influence of glutathione fructosylation on its properties Mikhail Linetsky * , Ekaterina V. Shipova, Ognyan K. Argirov Mason Eye Institute, University of Missouri, Columbia, MO 65201, USA Received 2 January 2006, and in revised form 6 February 2006 Available online 13 March 2006 Abstract Incubation of fructose and glutathione leads to the formation of N-2-deoxy-glucos-2-yl glutathione as the major glycation product, with characteristic positive ion at 470 Th in LC–MS spectra. Glutathione disulfide and fructose generate two compounds: N-2-deoxy- glucos-2-yl glutathione disulfide (m/z = 775 Th) and bis di-N,N 0 -2-deoxy-glucos-2-yl glutathione disulfide (m/z = 937 Th). N-2-deoxy- glucos-2-yl glutathione is 2.5-fold less effective than glutathione in reducing dehydroascorbic acid. Glutathione peroxidase and glutahi- one-S-transferase exhibit marginal activity toward N-2-deoxy-glucos-2-yl glutathione, while glyoxalase I shows 44.9% of the enzyme’s specific activity. Glutathione reductase demonstrates 6.9% of the enzyme’s specific activity with bis di-N,N 0 -2-deoxy-glucos-2-yl gluta- thione, while with mono-N-glucosyl glutathione disulfide retained 5 6.1% of the original activity. Glutathione reductase could not reduce N-2-deoxy-glucos-2-yl glutathione in mixed disulfide with cS-crystallin, but reduced glutathione in mixed disulfide with cS-crystallin by 90%. The presence of N-2-deoxy-glucos-2-yl glutathione in mixed disulfide with cS-crystallin makes this molecule more susceptible to unfolding than native cS-crystallin. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Non-enzymatic glycation; Fructose; Glutathione; Glutathione mixed disulfides Glutathione (GSH) 1 is essential for maintaining sulfhy- dryl groups of enzymes and lens proteins, thus preventing the formation of protein–protein disulfides [1–3]. It also keeps ascorbic acid in the reduced state and detoxifies oxy- gen-free radicals and xenobiotics [4]. In the newborn human lens, GSH levels reach 6.0 mM [5]. However, in the presence of persistent hyperglycemia, as seen in diabe- tes, a precipitous drop in GSH levels is the most common and earliest biochemical change, even before cataract develops [6–9]. Major factors thought to be involved in dia- betes-related cataract formation include GSH oxidation by oxygen-derived species in combination with the inability of glutathione reductase (GR) to maintain the pool of reduced GSH [8], decreased levels of GSH-synthesizing enzymes [10], osmotic stress caused by intralenticular accu- mulation of polyols mediated by aldose reductase [8,11,12] and leakage of oxidized GSH from diabetic lens [6,8,10– 12]. Conversely, Obrosova et al. [8,11] showed that while GSH levels dropped almost 5-fold in mildly diabetic rat lens, oxidized GSH levels remained unchanged. Other stud- ies have demonstrated increased GR activity in diabetic lenses, both in humans [7] and in rats [8,11]. However, no significant change in the NADP + /NADPH ratio has been observed, which argues against loss of GR as a cause of GSH depletion [8]. Significant leakage of oxidized GSSG has not been found in diabetic rat lenses [8]. Together, these results may indicate that oxidative stress does not play a significant role in GSH depletion in diabetic lens. Similar results were obtained in the mouse diabetic model www.elsevier.com/locate/yabbi Archives of Biochemistry and Biophysics 449 (2006) 34–46 ABB 0003-9861/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.02.019 * Corresponding author. Fax: +1 573 884 4868. E-mail address: LinetskyM@health.missouri.edu (M. Linetsky). 1 Abbreviations used: GSH, glutathione (reduced form); GSSG, gluta- thione (oxidized form); Fru, fructose; N-2-deoxy-glucos-2-yl glutathione; Glc-GSH, N-2-deoxy-glucos-2-yl glutathione disulfide; Glc-GSSG, bis di-N,N 0 -(2-deoxy-D-glucos-2-yl)-glutathione disulfide; Glc-GSSG-Glc, GR-glutathione reductase; GPx, glutathione peroxidase; GST, glutathi- one-S-transferase; GO1, glyoxalase I; CDNB, 1-chloro-2,4-dinitroben- zene; GLC-D, () glucose; TCEP, tricarboxyethyl phosphine; TCA, trichloroacetic acid; TMA, trimethylamine; DTNB, 5,5 0 -dithiobis (2- nitrobenzoic acid); TNBS, 2,4,6-trinitrobezene sulfonic acid; ROS, reactive oxygen species; cS-S-SG, mixed disulfide of GSH and cS- crystallin; cS-S-SG-Glc, mixed disulfide of N-2-deoxy-glucos-2-yl gluta- thione and cS-crystallin.