Inactivation and modification of superoxide dismutase by glyoxal: Prevention by antibodies Rukhsana Jabeen a,b , M. Saleemuddin b , John Petersen a , Amin Mohammad a, * a Department of Pathology, University of Texas Medical Branch, 301, University Boulevard, CSB 500, Galveston, TX 77555-0551, USA b Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India Received 3 May 2006; accepted 26 October 2006 Available online 27 November 2006 Abstract Glyoxal is an endogenous compound, the levels of which are increased in various pathologies associated with hyperglycaemia and other related disorders. It has been reported to inactivate critical cellular enzymes by promoting their cross-linking and perpetuates advanced glycation end-product (AGE) formation. In this study, we used superoxide dismutase (SOD) as a model to investigate the ability of specific anti-enzyme antibodies and monomer Fab fragments to protect against glyoxal-induced deactivation and aggregate formation. We found that glyoxal deac- tivated SOD, in a concentration and time-dependent fashion. The enzymatic activity was monitored spectrophotometrically and it was found that enzyme lost approximately 95% of its original activity, when exposed to 10 mM glyoxal for 120 h. SDS-polyacrylamide gel electrophoresis demonstrated the formation of high molecular weight aggregates in SOD samples exposed to glyoxal. Surface-enhanced laser desorption/ion- ization time of flight mass spectrometry (SELDI-TOF-MS) showed increase in relative molecular mass (M r ), upon exposure to glyoxal. Specific anti-enzyme antibodies and monomer Fab fragments markedly inhibited SOD deactivation caused by glyoxal and decreased the extent of cross- linking or formation of aggregates. This protection by the antibodies or Fab fragments was specific since, other non-specific antibodies were not able to protect SOD. Previously, antibodies have been used to prevent aggregation of b-amyloid peptides in Alzheimer and prion-protein disease. Our findings provide a new perspective, for use of antibodies to prevent the biomolecules against glycation-induced deactivation and alteration. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Glyoxal; Maillard reaction; SOD; Polyclonal antibodies 1. Introduction Post-translational non-enzymatic glycation of proteins rep- resents a complex network of reactions which are believed to play an important role in the development of physiological and pathophysiological processes, such as aging, diabetes, athero- sclerosis, neurodegenerative diseases, vascular diseases and chronic renal failure [1,2]. Non-enzymatic glycation also termed as Maillard reaction is a spontaneous condensation reaction that spontaneously occurs between the aldehyde groups of free unbound sugars in the open chain form and the free amino groups of proteins, lipids and nucleic acids. This results in the formation of a labile Schiff base. The Schiff base being highly prone to oxidation and free radical generation, rearranges to form a more stable Amadori product [3,4]. The Amadori compounds are slowly degraded, in complex reaction pathways via dicarbonyl intermediates, to a number of com- pounds, designated collectively as advanced glycation end- products (AGEs) or Maillard reaction products (MRPs) [5,6]. The post-Amadori products include highly reactive a-dicarbonyl intermediates like glyoxal, Amadori dione, 3-deoxyglucosone and methylglyoxal [7]. Glyoxal is particularly interesting because it originates from pathways that have been linked to various pathologies including sugar autoxidation, DNA Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacryamide gel electrophoresis; PMS, phenazine methosulphate; NBT, nitroblue tetrazolium; SOD, superoxide dismutase; AGE, advanced glycation end-product; CML, carboxy(methyl lysine); CMA, carboxy methyl arginine; PLGA, poly lac- tide-co-glycolic acid. * Corresponding author. Tel.: þ1 409 7722527; fax: þ1 409 7725683. E-mail address: rujabeen@utmb.edu (A. Mohammad). 0300-9084/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2006.10.015 Biochimie 89 (2007) 311e318 www.elsevier.com/locate/biochi