A procedure for the rapid screening of Maillard reaction inhibitors Shamila Fatima a , Deeba S. Jairajpuri a , M. Saleemuddin a,b, ⁎ a Department of Biochemistry, Aligarh Muslim University, Aligarh 202002, India b Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India Received 6 July 2007; received in revised form 18 October 2007; accepted 22 October 2007 Abstract A procedure for the rapid screening of inhibitors of glycation reaction, based on their ability to protect RNase against sugar induced inactivation of the enzyme is described. Glycation is implicated in variety of disorders including diabetes, atherosclerosis various micropathies yet is a slow process both in vivo and in vitro. In order to speed up glycation, the reaction was carried out at 60 °C using a thermostable protein RNase and ribose, a sugar that is known to react rapidly than glucose in the glycation reaction. It was observed that incubation of RNase with ribose at 60 °C in rapid inactivation of the enzyme with a parallel decrease in tyrosine fluorescence, enhancement in new fluorescence and hyperchromicity in the UV-region. No such alterations in the enzyme activity were observed when the incubation was carried out in absence of the sugar. Compounds and drugs that are known to act as inhibitors of glycation reaction restricted the ribose-induced inactivation of RNase. RNase immobilized on CNBr-activated Sepharose was also sensitive to exposure to ribose and appeared a better system to screen inhibitors of glycation from natural sources that contain substances that interfere with the assay of enzyme as well as in the study of post Amadori inhibitors of glycation. © 2007 Elsevier B.V. All rights reserved. Keywords: Glycation inhibitors; RNase; Maillard reaction; Ribose 1. Introduction During Maillard reaction reducing sugars react with amino groups of biomolecules including proteins, lipids, nucleic acids to form Schiff base which in turn undergoes transformation to a variety of advanced glycation end products (AGEs) [1,2]. The in vivo significance of Maillard reaction was first recognized with the detection of elevated levels of hemoglobin in the diabetic blood [3]. Subsequent evidence suggested that the AGE may play an important role in the etiology of variety of diabetic complications [4] and aging [5,6]. AGEs have also been im- plicated in a variety of disorders including inflammation [7], atherosclerosis [8], neurodegenerative disorders [9] and cancer [10]. Glycated protein may lose biological activity, turn toxic, develop new antigenic determinants and damage cell in a va- riety of ways after uptake mediated by specific receptors present on the surface of several cells [11,12]. Several lines of evidence suggest that agents that restrict AGE formation result in reduction of various diabetic com- plications including retinopathy [13,14] and can be considered as diabetic complication drugs. While several other strategies also appear promising [15–17], current emphasis is on the substances that act inhibitors of AGE formation. Among the compounds evaluated as AGE inhibitors, aminoguanidine (AG) [18], aspirin [19], vitamin B 6 [20] taurine [21] and quercitin [22] are important. Among these AG is in late stages of clinical trials, yet the effective dose of aminoguanidine is quite high with accompanying risks of side effects. In search of alternative drugs, screening of natural sources for potential glycation in- hibitors has also been undertaken [22]. The standard proce- dures of assay of glycation require sophisticated equipment Available online at www.sciencedirect.com J. Biochem. Biophys. Methods 70 (2008) 958 – 965 www.elsevier.com/locate/jbbm Abbreviations: AGE, advanced Glycation end products; DETAPAC, diethylenetriaminepenta acetic acid; EDTA, ethylenediaminetetraacetic acid; AG, aminoguanidine; RNA, ribonucleic acid; PAGE, polyacrylamide gel electrophoresis; CNBr, cynogen bromide; RNase A, ribonuclease A; SDS, sodium dodecyl sulphate. ⁎ Corresponding author. Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India. Tel.: +91 571 2700741; fax: +91 571 2721776. E-mail address: msaleemuddin47@gmail.com (M. Saleemuddin). 0165-022X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2007.10.002