Oxidation of cystatin imparted by riboflavin generated free radicals: Spectral analysis.

Thiol Protease inhibitors (cystatins) are endogenous natural inhibitors of cysteine proteases. They are present in all mammalians cells and body fluids. Cystatin are allocated into three major families. Family -I stefins, family -II cystatins and family -III kininogens, according to their amino acid sequence, molecular weight, carbohydrate content and disulphide bonds. It has been investigated that thiol proteases (cathepsin) and their endogenous inhibitor, cystatins have been closely associated with diseases like Alzheimer's, Prions, neurodegenerative diseases, cancer and diabetes. Photodynamic effect of various sensitizers' have long been applied to delineate structural and functional properties of biologically active proteins. Flavins are well known to photo oxidize amino acids which effects conformation of proteins. Riboflavin (Vit B2 ) with a recommended daily requirement of approximately 2-3 mg is a yellow pigment, It is widely distributed in human tissues and blood, in both free and conjugated forms. In the present Study it has been shown that cystatin purified from buffalo brain (BC) is susceptible to reactive oxygen species generated by photo activation of riboflavin. It was observed that Photo activated riboflavin leads to inactivation of BC. Major Loss of tryptophan intensity was observed in the presence of purified thiol protease inhibitor upon incubation with 50 μM of riboflavin. In order to inspect the type of reactive oxygen species involved in inactivation of the inhibitor, different scavenger's were used namely glucose, potassium Iodide, sodium azide, manitol, thiourea, sodium benzoate, curcumin, quercetin, ascorbic acid and uric acid. It was found that Glucose, Potassium Iodide and sodium azide, have preventive effect on photo inactivation of the purified cystatin whilst other scavengers illustrated diminutive defensive effect.