Colloids and Surfaces B: Biointerfaces 125 (2015) 170–180 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces jo ur nal ho me p ag e: www.elsevier.com/locate/colsurfb The impact of calcium ion on structure and aggregation propensity of peroxynitrite-modified lens crystallins: New insights into the pathogenesis of cataract disorders Maryam Ghahramani a , Reza Yousefi a,∗ , Kazem Khoshaman a , Mohammad-Mehdi Alavianmehr b a Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran b Department of Chemistry, Shiraz University of Technology, Shiraz, Iran a r t i c l e i n f o Article history: Received 26 August 2014 Received in revised form 30 September 2014 Accepted 3 November 2014 Available online 24 November 2014 Keywords: Peroxynitrite (PON) Calcium Lens crystallins Aggregation Cataract a b s t r a c t As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has been indicated in the pathogenesis of various ocular disorders. The PON induces mobilization of intra cellular calcium which plays an important function in structure and activity of lens proteins. Moreover, the amount of cal- cium increases to the pathogenic level in the cataractous lenses. The aim of this study was to assess the impact of calcium ion on structure and aggregation of PON-modified lens crystallins, using spec- troscopic techniques and gel mobility shift assay. The PON modification of lens proteins was confirmed with detection of the significantly increased quantity of carbonyl group, dityrosine, nitrotyrosine and nitrotryptophan. Moreover, the modified proteins indicated high levels of solvent exposed hydropho- bic surfaces and markedly elevated proteolytic instability which can be explained with their structural alteration upon this type of modification. The results of UV–vis absorption studies suggest that PON- modified lens crystallins are highly sensitive to aggregation in the presence of both physiological and pathological ranges of calcium ion. Also, the results of thioflavin T fluorescence study indicated absence of any ordered aggregate entity in the calcium-induced aggregate samples. The results of gel mobility shift assay demonstrated the importance of calcium ion in the induction of disulfide and dityrosine covalent cross-linking and formation of the oligomeric structure with relatively larger sizes in the PON-modified crystallins compared to the non-modified protein counterparts. Overall, this study may suggest that a simultaneous raise of calcium ion and PON in the eye ball is an important risk factor for development of cataract diseases. © 2014 Elsevier B.V. All rights reserved. 1. Introduction As a highly potent reactive oxygen and nitrogen species, per- oxynitrite (PON) is formed in a reaction between two free radicals, superoxide anion (O 2 •- ) and nitric oxide (NO • ) [1–3]. While super- oxide is a byproduct of normal metabolism, nitric oxide is produced by the activity of two constitutive nitric oxide synthases (nNOS and eNOS) and one inducible enzyme counterpart (iNOS) which can be stimulated with certain immunologic or inflammatory stimuli [4–7]. The rate of PON generation is significantly enhanced under pathological conditions such as inflammation and diabetes, where stimulated cells release NO • and O 2 •- at the elevated rates which favor formation of this oxidative agent [8–11]. PON which ∗ Corresponding author. Tel.: +98 7116137665; fax: +98 7112280916. E-mail address: ryousefi@shirazu.ac.ir (R. Yousefi). influences the inflammatory responses at multiple levels has been indicated to contribute in the pathogenesis of diabetic complica- tions [12,13]. The three NOS isoforms have been identified in dif- ferent parts of the eye [14,15]. Moreover, the induction of iNOS and overproduction of PON have been indicated in ocular inflamma- tory pathologies and in the retinas of human subject with diabetic retinopathy [14,16–18]. The enhanced formation of PON has been indicated in ocular fluid of various optical disorders such as uveitis, corneal damages, glaucoma, retinopathy, and senile cataract [19]. The pathway of PON pathobiology can be explained by the ability of this potent oxidizing agent to modify variety of biomolecules such as proteins, lipids, sugars, DNA and small antioxidant molecules, e.g., glutathione [2,20–24]. Moreover, PON demon- strates a very high affinity for Tyr, Trp and Cys residues in proteins [25–27]. Accordingly, various biomarkers of PON-induced protein modifications have been introduced as nitrotyrosine, dityrosine, nitrotryptophan, protein carbonyls, cysteine oxidation, protein http://dx.doi.org/10.1016/j.colsurfb.2014.11.002 0927-7765/© 2014 Elsevier B.V. All rights reserved.