Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.

BACKGROUND Diabetic retinopathy is becoming one of the common blinding disease in the world affecting people in both developing and developed countries.Basic mechanism thought to be of advanced glycation end products and othertoxic mediators causingtissue destruction and pathological process. Antioxidants have a major role in preventing this pathological process. Among various antioxidants some of the common blood products have thought to have a role. One among them is serum bilirubin. This study is done to know the correlation of diabetic retinopathy and serum bilirubin levels and thus know its importance in future in preventing progression of this blinding disease. METHODS A cross sectional study was done among OPD patients with type 2 diabetes for a period of one year. Inclusion criteria were diagnosed cases of type 2 diabetes for more than 1 year and age above 40 years. Exclusion criteria included all systemic diseases/drugs affecting liver function tests, confounding factors affecting serum bilirubin levels, extremely poor glycemic control and subjects in whom fundus was not visible due to media opacities excluding causes linked with diabetic retinopathy. After taking consent, detailed history and ophthalmic evaluation, venous blood was drawn and sent for serum bilirubin analysis. Diabetic retinopathy was classified according to ETDRS classification. Statistical study was done after compiling data. RESULTS Among the study subjects – 38.2% were diabetics. Common age group was 51 to 60 years with incidence of diabeties more in males 64.3%. Among diabetic retinopathy noted– mild NPDR was 31%, moderate NPDR was 35.7%, severe NPDR was 11.9%, very severe NPDR was 4.8% and PDR was 16.6 % respectively. The mean serum total bilirubin levels in non DR was 0.597 ± 0.17, mild NPDR was 0.4 ± 0. 15, moderate NPDR was 0.36 ±0.12, severe NPDR was 0.36±0.17, very severe NPDR was 0.35±0.07, low risk PDR was 0.3±0.10 and high risk PDR was 0.32±0.15 respectively. CONCLUSIONS This study concluded that severity of diabetic retinopathy was inversely proportional to the total, direct and indirect serum bilirubin levels. KEY WORDS Diabetic Retinopathy, Serum Bilirubin, Diabetes Mellitus, ETDRS