In vitro biochemical characterization and genotoxicity assessment of Sapindus saponaria seed extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Sapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported. AIM OF THE STUDY: Plant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases. MATERIALS AND METHODS: Protein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically. RESULTS: Seed proteins extract of S. saponaria was evaluated until 100 µg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ​​the biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects. CONCLUSION: The overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs.

Insight into the biochemical characterization of phytocystatin from Glycine max and its interaction with Cd+2 and Ni+2.

Phytocystatins are cysteine proteinase inhibitors ubiquitously present in plants and animals. They are known to carry out various significant physiological functions and also maintain the balance of protease-antiprotease activity. In the present disquisition, a phytocystatin after preliminary treatment has been isolated and purified to homogeneity from soybean (Glycine max) by a simple two-step stratagem using ammonium sulfate fractionation and gel filtration chromatography performed on Sephacryl S-100-HR. Soybean phytocystatin (SBPC) was purified with a fold purification of 635 and percent yield of 77.6%. A single band was observed on native gel electrophoresis confirming the homogeneity of the purified SBPC. The molecular weight of SBPC was found to be 19.05 kDa as determined by SDS-PAGE. The SBPC was found to be devoid of carbohydrate moieties and sulfhydryl group content. The binding stoichiometry of SBPC-papain interaction was determined by isothermal calorimetry suggesting 1:1 complex, and the value of binding constant (K) was found to be 2.78 × 105  M-1 The affinity of binding (Kd ) value obtained through ITC was 3.59 × 10-6  M. The purified SBPC was found to be stable in the pH range of 3 to 7 and is thermostable up to 50°C. The UV-visible and fluorescence studies showed significant changes in the conformation upon the formation of the SBPC-papain complex. Furthermore, fluorescence spectroscopy, ANS binding, and caseinolytic activity assay were conducted out to explore the effect of metal ions on SBPC which showed that there was a loss in the inhibitory activity along with conformational changes of SBPC upon complex formation with Cd+2 and Ni+2 .

Role of phytocystatin in combating metal ion induced conformational alterations in glutathione reductase.

Glutathione reductase (GR) is a flavoprotein that catalyses the reduction of oxidized glutathione (GSSG) to reduced glutathione (2GSH) in the presence of coenzyme NADPH. The importance of glutathione stems from the fact that it serves an important role in various metabolic processes. Plants growing in highly polluted areas are exposed to higher concentration of metal ions; thereby feeling abiotic stress and affecting various regulatory enzyme activities. In this study, effect of metal ions has been studied on GR. Phytocystatins show an increased expression in abiotic stress conditions. Here in, the effect of cystatin isolated from yellow mustard seeds (YMP) on heavy metals induced conformational changes in GR was investigated making use of GR activity assay, UV-absorption spectroscopy, fluorescence spectroscopy, FTIR, CD, ITC and SEM analysis. The results obtained clearly reveals that metal ions like Cu2+ and Zn+2 induces concentration dependent conformational changes in GR; YMP restores these alterations in way decreasing the effective concentration of metal ions.

Purification and characterization of a cystatin like thiol protease inhibitor from Brassica nigra.

Phytocystatins or plant cystatins belong to a group of thiol protease inhibitors present ubiquitously in living system. They play a crucial role in cellular protein turnover thereby showing involvement in a wide array of physiological processes in plants. With wide importance and tremendous potential applications in the fields of genetic engineering, medicine, agriculture, and food technology, it is imperative to identify and isolate such protease inhibitors from different cheap and easily available plant sources. Present study focuses on the isolation, purification and characterization of a cystatin like thiol protease inhibitor from the seeds of Brassica nigra (rai mustard) following a simple two-step method using ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with 51.85% yield and 151.50 fold purification. Rai seed cystatin (RSC) gave a molecular mass of ~19.50 kDa as determined by SDS PAGE and gel filtration behaviour. Stokes radius and diffusion coefficient of RSC were 19.80 Šand 11.21 × 10-7 cm2 s-1 respectively. Kinetic analysis revealed a reversible and non-competitive mode of inhibition with RSC showing highest inhibition towards papain (Ki = 1.62 × 10-7 M) followed by ficin and bromelain. Purified RSC possessed an α helical content of 35.29% as observed by far-UV CD spectroscopy. UV, fluorescence, CD and FTIR spectral studies revealed a significant conformational alteration in one or both the proteins upon RSC-papain complex formation. Isothermal Titration Calorimetry (ITC) analysis further revealed the values for different thermodynamic parameters involved in complex formation, indicating the process to be enthalpically as well as entropically driven with forces involved in binding the proteins to be electrostatic in nature. Additionally binding stoichiometry (N) of 0.95 ±â€¯0.08 sites indicates that each molecule of RSC is surrounded by nearly one papain molecule.

The Anti-Angiogenic Activity of a Cystatin F Homologue from the Buccal Glands of Lampetra morii.

Cystatins are a family of cysteine protease inhibitors which are associated with a variety of physiological and pathological processes in vivo. In the present study, the cDNA sequence of a cystatin F homologue called Lm-cystatin F was cloned from the buccal glands of Lampetra morii. Although Lm-cystatin F shares a lower homology with cystatin superfamily members, it is also composed of a signal peptide and three highly conserved motifs, including the G in the N-terminal, QXVXG, as well as the PW in the C-terminal of the sequence. After sequence optimization and recombination, the recombinant protein was expressed as a soluble protein in Escherichia coli with a molecular weight of 19.85 kDa. Through affinity chromatography and mass spectrometry analysis, the purified protein was identified as a recombinant Lm-cystatin F (rLm-cystatin F). Additionally, rLm-cystatin F could inhibit the activity of papain. Based on MTT assay, rLm-cystatin F inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) dose dependently with an IC50 of 5 µM. In vitro studies show that rLm-cystatin F suppressed the adhesion, migration, invasion, and tube formation of HUVECs, suggesting that rLm-cystatin F possesses anti-angiogenic activity, which provides information on the feeding mechanisms of Lampetra morii and insights into the application of rLm-cystatin F as a potential drug in the future.

An Immunosuppressive Tick Salivary Gland Protein DsCystatin Interferes With Toll-Like Receptor Signaling by Downregulating TRAF6.

Ticks, blood-feeding arthropods, and secrete immunosuppressive molecules that inhibit host immune responses and provide survival advantages to pathogens. In this study, we characterized the immunosuppressive function of a novel tick salivary protein, DsCystatin, from Dermacentor silvarum of China. DsCystatin directly interacted with human Cathepsins L and B and inhibited their enzymatic activities. DsCystatin impaired the expression of inflammatory cytokines such as IL1ß, IFNγ, TNFα, and IL6 from mouse bone marrow-derived macrophages (BMDMs) that had been stimulated with LPS or Borrelia burgdorferi. Consistently, DsCystatin inhibited the activation of mouse BMDMs and bone marrow-derived dendritic cells by downregulating the surface expression of CD80 and CD86. Mechanically, DsCystatin inhibited LPS- or B. burgdorferi-induced NFκB activation. For the first time, we identified that DsCystatin-attenuated TLR4 signaling by targeting TRAF6. DsCystatin enhanced LPS-induced autophagy, mediated TRAF6 degradation via an autophagy dependent manner, thereby impeded the downstream phosphorylation of IκBα and the nuclear transport of NFκB. Finally, DsCystatin relieved the joint inflammation in B. burgdorferi or complete Freund's adjuvant induced mouse arthritis models. These data suggested that DsCystatin is a novel immunosuppressive protein and can potentially be used in the treatment of inflammatory diseases.

Insight into the functional and structural transition of garlic phytocystatin induced by urea and guanidine hydrochloride: A comparative biophysical study.

Cysteine proteinase inhibitors play an essential role in maintaining the proper functioning of all living cells by virtue of its thiol protease regulatory properties. Chemical denaturation of a new variant of cystatin super family has been studied by various biophysical techniques in order to characterize the unfolded and denatured state. Denaturation of garlic phytocystatin (GPC) has been investigated using urea and guanidine hydrochloride (GdnHCl). Different biophysical techniques such as intrinsic fluorescence, circular dichroism and FTIR exhibited an altered structure of garlic phytocystatin with increasing concentration of denaturant. The inhibitory activity of GPC decreases with increasing concentration of denaturant. Increased fluorescence intensity along with red shift reflects the unfolding of GPC at higher concentration of denaturant. GdnHCl induced unfolding showed presence of indiscernible intermediate as followed by ANS binding studies. However, denaturation by urea did not show any intermediates. Mid-point transition was observed at 4.7±0.1M urea and 2.32±0.1M GdnHCl. Circular dichroism and FTIR results indicate the 50% loss of secondary structure at 5M urea and 2.5M GdnHCl. This study provides intriguing insight into the possible alteration of structure, stability and function of GPC induced by urea and GdnHCl.

Isotope-coded protein label based quantitative proteomic analysis reveals significant up-regulation of apolipoprotein A1 and ovotransferrin in the myopic chick vitreous.

This study used isotope-coded protein label (ICPL) quantitative proteomics and bioinformatics analysis to examine changes in vitreous protein content and associated pathways during lens-induced eye growth. First, the vitreous protein profile of normal 7-day old chicks was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry. A total of 341 unique proteins were identified. Next, myopia and hyperopia were induced in the same chick by attaching -10D lenses to the right eye and +10D lenses to the left eye, for 3 and 7 days. Protein expression in lens-induced ametropic eyes was analyzed using the ICPL approach coupled to LCMS. Four proteins (cystatin, apolipoprotein A1, ovotransferrin, and purpurin) were significantly up-regulated in the vitreous after 3 days of wearing -10D lenses relative to +10D lens contralateral eyes. The differences in protein expression were less pronounced after 7 days when the eyes approached full compensation. In a different group of chicks, western blot confirmed the up-regulation of apolipoprotein A1 and ovotransferrin in the myopic vitreous relative to both contralateral lens-free eyes and hyperopic eyes in separate animals wearing +10D lenses. Bioinformatics analysis suggested oxidative stress and lipid metabolism as pathways involved in compensated ocular elongation.

Modulation of goat monocyte function by HCcyst-2, a secreted cystatin from Haemonchus contortus.

Modulation and suppression of the host immune response by nematode parasites have been reported extensively and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulator. In the present study, we cloned and produced recombinant cystatin protein from nematode parasite Haemonchus contortus (rHCcyst-2) and investigated its immunomodulatory effects on goat monocyte. rHCcyst-2 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B and papain. Immunohistochemical test demonstrated that the native HCcyst-2 protein was predominantly localized at the body surface and internal surface of the worm's gut. We demonstrated that rHCcyst-2 could be distinguished by antisera from goats experimentally infected with H. contortus and could uptake by goat monocytes. The immunomodulatory effects of HCcyst-2 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-2 with goat monocytes. The results showed that the interaction of rHCcyst-2 decreased the production of TNF-α, IL-1ß and IL-12p40. However, it significantly increased the secretion of IL-10 in goat monocytes. After rHCcyst-2 exposure, the expression of MHC-II on goat monocytes was inhibited. Moreover, rHCcyst-2 could up-regulate the LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-2 inhibited the phagocytosis of goat monocytes. Our findings provided potential target as immunoregulator, and will be helpful to illustrate the molecular basis of host-parasite interactions and search for new potential molecule as vaccine and drug target candidate.

Biochemical, immunological and kinetic characterization and partial sequence analysis of a thiol proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders.

In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10-4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus.

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10-7 cm2 s-1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (Ki = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters - ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (-5.78 kcal mol-1) and positive ΔS (11.01 cal mol-1 deg-1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (-9.19 kcal mol-1) value implies a spontaneous CBC-papain interaction.

Deciphering the interaction of bovine heart cystatin with ZnO nanoparticles: Spectroscopic and thermodynamic approach.

ZnO-NPs have been widely used in biomedical fields such as therapeutics, cellular imaging, and drug delivery. However, the risk of exposure of nanoparticles to the biological system is not well understood. Nanoparticle-protein interaction is pivotal to understand their biological behavior and predict nanoparticle toxicity that is crucial for its safer applications. In the present study zinc oxide nanoparticles (ZnO-NPs) were synthesized and subjected to interact with buffalo heart cystatin (BHC), purified from buffalo heart, to assess the effect(s) of ZnO-NPs on the structure and function of BHC. In vitro toxicity assessments revealed that BHC, upon interaction with ZnO-NPs, led to the altered protein conformation and perturbed function. A decrease in the anti-papain activity of BHC was observed. Spectroscopic studies demonstrated that formation of BHC-ZnO-NPs complex accompanied by structural changes in BHC along with a significant decrease in its α-helical content. ITC determined the thermodynamic parameters of binding between ZnO-NPs and BHC quantitatively. Increased surface hydrophobicity (change in the tertiary structure) was observed by ANS fluorescence that demonstrated the formation of molten globular intermediates that were found to be stable without any signs of aggregation as depicted by ThT fluorescence. TEM images gave the physical evidence of the formation of ZnO-NPs-BHC corona.

Structural transition of kidney cystatin induced by silicon dioxide nanoparticles: An implication for renal diseases.

Nanotechnology is one of the fastest growing fields of science owing to use of nanomaterials in industries and medicine across the globe. Currently silicon dioxide nanoparticles (SiO2 NPs) are one of the most popular nanomaterials owing to their inert toxicity profile and hence exposure to SiO2 nanoparticles is on the increase. Cystatins are thiol proteinase inhibitors (TPIs) ubiquitously distributed in plants and animals and they are now at the heed of a number of normal and pathological conditions and shouldn't be regarded solely as TPIs. Up till now many studies have targeted the potential toxicity of NPs on pulmonary target; although little focus is given to kidney which is a secondary target organ. The objective of this work is to study the structural changes in buffalo kidney cystatin (BKC) induced by SiO2 NPs. UV and Fluorescence spectroscopy shows BKC transformation from native to non-native form evident by decreased absorbance and increased fluorescence. FTIR and CD spectroscopy further confirmed secondary structure disruption of BKC. Isothermal titration calorimetry (ITC) and microscopy were resorted to visualize interaction between SiO2 NPs and BKC. Comet assay and MTT assay were utilized to perceive the toxicity of SiO2 NPs incubated BKC; decreased cell viability clearly suggesting toxicity of SiO2 NPs incubated BKC. Our work suggests that SiO2 NPs have a deteriorating effect on BKC thereby causing a decrease in its ability to inhibit papain and hence less functionality. This study also shows that BKC transforms to a toxic non-native form in presence of SiO2 NPs.

Functional proteomics-aided selection of protease inhibitors for herbivore insect control.

Studies have reported the potential of protease inhibitors to engineer insect resistance in transgenic plants but the general usefulness of this approach in crop protection still remains to be established. Insects have evolved strategies to cope with dietary protease inhibitors, such as the use of proteases recalcitrant to inhibition, that often make the selection of effective inhibitors very challenging. Here, we used a functional proteomics approach for the 'capture' of Cys protease targets in crude protein extracts as a tool to identify promising cystatins for plant improvement. Two cystatins found to differ in their efficiency to capture Cys proteases of the coleopteran pest Leptinotarsa decemlineata also differed in their usefulness to produce transgenic potato lines resistant to this insect. Plants expressing the most potent cystatin at high level had a strong repressing effect on larval growth and leaf intake, while plants expressing the weakest cystatin showed no effect on both two parameters compared to untransformed parental line used for genetic transformation. Our data underline the relevance of considering the whole range of possible protease targets when selecting an inhibitor for plant pest control. They also confirm the feasibility of developing cystatin-expressing transgenics resistant to a major pest of potato.

Physico-chemical and in-silico analysis of a phytocystatin purified from Brassica juncea cultivar RoAgro 5444.

This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3-10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% ß-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain-phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.

Purification and biochemical characterization of phytocystatin from Brassica alba.

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two-step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S-100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180-fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10(-7) cm(2) s(-1) respectively. The isolated phytocystatin was found to be stable in the pH range of 6-8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non-competitive type of inhibition and inhibited papain more efficiently (K(i) = 3 × 10(-7) M) than ficin (K(i) = 6.6 × 10(-7) M) and bromelain (K(i) = 7.7 × 10(-7) M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content.

Phytocystatins and their potential to control plant diseases caused by fungi.

Plant cystatins, also called phytocystatins, constitute a family of specific cysteine protease inhibitors found in several monocots and dicots, where they can be involved in the regulation of several endogenous processes and in defense against pests and pathogens, as well as in response to abiotic stress. In this mini-review we aimed to present isolated and characterized phytocystatins with potential use in control of plant disease caused by fungi.

Expression and purification of soluble porcine cystatin 11 in Pichia pastoris.

Cystatin 11 (CST11) belongs to the cystatin type 2 family of cysteine protease inhibitors and exhibits antimicrobial activity in vitro. In this study, we describe the expression and purification of recombinant porcine CST11 in the Pichia pastoris system. We then assess its antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis by liquid growth inhibition assay. Kinetic studies indicate that the recombinant porcine CST11 has high potency against E. coli and S. aureus. Scanning electronic microscope analysis showed that CST11 might be targeting the bacterial membrane and, thus, could potentially be developed as a therapeutic agent for inhibiting microbe infection without the risk of antibiotic resistance.

An improved method to extract and purify cystatin from hen egg white.

Hen egg white cystatin, an inhibitor of cysteine proteinase, may have wide applications for improving human health. However, its pricy cost associated with extraction and preparation has hurdled its further utilization. The objective was to develop an improved method to extract and purify cystatin from egg white. After removal of ovomucin, a fraction containing cystatin was obtained by cation exchange chromatography, and further purified by affinity chromatography using a cm-papain-Sepharose column. The prepared cystatin was then characterized by SDS-PAGE, Western-Blot, and LC-MS/MS, and its purity was determined by HPLC method instead of the conventional immunodiffusion method. The protein content of cystatin extract was 66.4 ± 2.3%. In comparison with the conventional method, the purity of cystatin was improved from 56.6 ± 1.7% to 93.3 ± 4.0%, and its yield was improved from 21.3 ± 1.2% to 33.6 ± 1.5%. Relative activities of cystatin to inhibit papain prepared by our method and the conventional method were determined to be 88 ± 7% and 91 ± 4% respectively, against a cystatin standard from Sigma. This suggested no significant loss of activity during the separation process.

Effect of non-enzymatic glycation on cystatin: a spectroscopic study.

The study shows the effect of nonenzymatic glycation on conformation and inhibitory activity of chick pea cystatin (CPC). CPC was incubated with different reducing sugars, pentose (D-Ribose), hexoses (D-Glucose, D-Fructose) at 37 °C for 5 weeks. To evaluate the modification of CPC by these different sugars during the glycation process the extent of the Maillard reaction, conformational, structural and functional changes were investigated. The behaviour of glycated CPC was monitored by the techniques of UV and fluorescence spectroscopies. Specific fluorescence was employed to characterise the glycation and AGEs. The anti-papain activity of glycated CPC was found to be significantly lower as compared to its non-glycated form. Glycation with ribose led to maximum loss in inhibitory activity. It was found that the incubation of CPC with all the mentioned sugars led to a parallel increase in tryptophan fluorescence as well as in Maillard and other AGEs specific fluorescence values and hyperchromicity in the UV-region. Among the sugars studied comparatively ribose was found to be the most active in inducing structural and conformational alterations in the protein suggesting its high reactivity with protein amino groups.