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

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

Anna Feroz, Peerzada Shariq Shaheen Khaki, Azad Alam Siddiqui, Fakhra Amin, Mohd Sajid Khan, Bilqees Bano PII: DOI: Reference:

S0141-8130(18)34215-6 https://doi.org/10.1016/j.ijbiomac.2018.12.169 BIOMAC 11328

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

12 August 2018 17 December 2018 18 December 2018

Please cite this article as: Anna Feroz, Peerzada Shariq Shaheen Khaki, Azad Alam Siddiqui, Fakhra Amin, Mohd Sajid Khan, Bilqees Bano , Purification and characterization of a cystatin like thiol protease inhibitor from Brassica nigra. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.12.169

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ACCEPTED MANUSCRIPT Purification and characterization of a cystatin like thiol protease inhibitor from Brassica nigra Anna Feroz1,2, Peerzada Shariq Shaheen Khaki1, Azad Alam Siddiqui1, Fakhra Amin3, Mohd Sajid Khan2, Bilqees Bano1 1

Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh, U.P. India 202002

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Department of Zoology, Faculty of Life Sciences, AMU, Aligarh, U.P. India 202002

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Corresponding author:

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Nanomedicine & Nanobiotechnology Lab, Department of Biosciences, Integral University, Lucknow, U.P. India 226026

Professor Bilqees Bano

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Department of Biochemistry Faculty of Life Sciences AMU, Aligarh U.P. 202002, India

Email: [email protected] [email protected] +91 - 9997 44 8621

ACCEPTED MANUSCRIPT ABSTRACT 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,

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and food technology, it is imperative to identify and isolate such protease inhibitors from

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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

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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

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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

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diffusion coefficient of RSC were 19.80 Å and 11.21× 10-7 cm2 s-1 respectively. Kinetic analysis revealed a reversible and non-competitive mode of inhibition with RSC showing highest

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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,

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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

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(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

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forces involved in binding the proteins to be electrostatic in nature. Additionally binding stoichiometry (N) of 0.95 ± 0.08 sites indicates that each molecule of RSC is surrounded by

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nearly one papain molecule.

Key Words: Brassica nigra, Phytocystatin, Purification, Rai seed cystatin, Thiol protease inhibitor, Kinetics, Spectroscopy, Isothermal Titration Calorimetry

Abbreviations: RSC – Rai Seed Cystatin; EDTA – Ethylene diamine tetra acetic acid; kDa – Kilo Dalton; CBB - Coomassie brilliant blue; UV – Ultraviolet; CD – Circular Dichroism; FTIR - Fourier transform infrared spectroscopy; ITC - Isothermal Titration Calorimetry

ACCEPTED MANUSCRIPT INTRODUCTION Cystatins are the most widely distributed class of thiol protease inhibitors present in the living system, comprising of a superfamily, members (sub families) of which include family I - the Stefins, family II - the Cystatins and family III - the Kininogens, classified on the basis of molecular complexity and biological distribution [1]. These ubiquitous inhibitors are important

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for the survival and well being of the living system and act as the regulators of protein turnover,

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any imbalance in which can lead to deleterious consequences [1,2]. Inhibitors of cysteine

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proteases in plant system, termed as phytocystatins, have emerged as an independent subfamily on the cystatin phylogenetic tree [3,4] and have been the object of intense research since their identification in plants by Soichi Arai and co-workers [2]. Like their mammalian counterparts,

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these plant protease inhibitors are known to form stoichiometric high affinity complexes with papain-like cysteine proteases and inhibit their hydrolytic activity reversibly in a competitive or

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non-competitive fashion [5].

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Presence of a tripartite wedge, common to all the cystatins, with three structural motifs possessing partially flexible N terminus (presenting a Gly residue) and two hairpin loops

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(carrying a conserved QxVxG motif and a Trp residue, respectively) confers the ability to these protease inhibitors to tightly bind and interact with the target proteases [2,6]. Besides the

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presence of tripartite wedge involved in the reaction mechanism, these plant based protease inhibitors are known to possess a unique consensus sequence ([LVI]-[AGT]-[RKE]-[FY]-[AS]-

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[VI]-x-[EDQV]-[HYFQ]-N) at the N-terminal α-helix region [6–8]. In addition comparative protein modelling shows the resemblance of some phytocystatins with family II cystatins of

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animal origin while others resemble that of family I cystatin of mammalian system. Further unlike the cystatins of animal origin and with the exception of Stefins, these plant based inhibitors are devoid of disulfide bonds and carbohydrate content [2,7,9,10]. So far phytocystatins have been identified from a variety of sources and among all, the best studied phytocystatin is the OC-I cystatin from rice (Oryza sativa) [7]. Further based upon the molecular complexity and sequence alignment, phytocystatins have been categorized into three simple groups with Group I comprising of small, low molecular mass proteins in the size range of 12kDa to 16kDa. Group II comprises of proteins with slightly higher molecular mass (23kDa)

ACCEPTED MANUSCRIPT along with a C-terminal extension presenting a SNSL motif [5] and Group III phytocystatins consist of high molecular mass proteins (85kDa) containing multiple cystatin domains and are found in potato and tomato [8,11]. From the functional point of view, phytocystatins are known to play a wide array of functions ranging from acting as an endogenous proteolysis regulator during seed development and

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maturation to the inhibition of extracellular cysteine proteases of herbivore arthropods, parasitic

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nematodes and microbial pathogens [2]. Further, reports suggest the down regulation of cystatin

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encoding genes (DC-CPIn) in senescent organs, correlated with a corresponding up-regulation of the gene (DC-CPIn) as observed in petals at the full blooming stage of flowers and in germinating storage organs [2,12]. Studies have also reported the up-regulation of cystatin

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mRNA transcripts in different plant tissues subjected to adverse environmental and biotic stress conditions such as drought [13–15], salinity [14–16], heat shock [14,16], cold shock [14,17,18],

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fungal infection [16], mechanical wounding [16] and insect herbivory [2,19]. It has also been observed that over expression of AtCYS1(cystatin from Arabidopsis thaliana) induced by

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wounding blocks cell death activated by either avirulent pathogens or by oxidative and

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nitrosative stress [20].

In synergism to the role played by phytocystatins, these protease inhibitors have found practical

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applicability and have been extensively explored for the pest management by increasing their expression in transgenic plants and it has been proposed that cystatins could synergistically

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supplement Bacillus thuringiensis (Bt) action in the effort to control Coleopteran insects and possibly delay build-up of Bt resistance in these insects [21]. Besides cystatins have also been

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known to act against fungal and viral pathogens by affecting their replication which require cysteine protease activity [21,22]. And as far the future prospects of cystatin engineering is concerned, a completely new application field might emerge with the basis on designing phytocystatin-based novel non-antibody scaffold protein, termed as Adhirons [23] which are highly specific binding reagents and have the potential to partly replace the applicability of antibodies in research, diagnostics and therapy [21]. Moreover engineered cystatin might present itself as a potent nutraceutical composite in a plant-derived ‘designer’ food product to effectively combat pathogen infection in the human digestive system [24] and may also act as a natural sweetener in the form of monellin [21,25].

ACCEPTED MANUSCRIPT Brassica, a genus of plants belonging to the mustard family (Brassicaceae), comprise a number of species of great economic value and has recently been the subject of intense research [26–30]. Rapeseed mustard (Brassica spp.), known for its oil seed production is the third important crop grown worldwide with the Indian subcontinent, Central Africa and parts of Russia being the principle growing regions [31]. The prime oil yielding varieties among various mustard species are Brassica juncea (B. juncea), and Brassica nigra (B.nigra). B. nigra commonly known as

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black mustard is the common oil seed crop known for its food flavouring properties and

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medicinal values [32–35]. With day to day increase in demand and less productivity due to the

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vulnerability of the crop to different biotic and abiotic stress conditions, it has become imperative to enhance the production by developing superior and stress resistant varieties of the

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crop while taking endogenous phytocystatin content of the crop into consideration. Keeping in view the plethora of knowledge, tremendous potential applications and futuristic

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scope, an attempt was made to identify, isolate and characterize a cystatin like thiol protease inhibitor from an unexplored rai mustard seed (Brassica nigra) variety, in terms of its

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physiochemical parameters, immunological properties, secondary structural content and kinetic specifications. Present study will not only give an insight in to the functional involvement of the

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purified cystatin type during various physiological and stress conditions in the rai mustard seeds but will also make additions to the already existing pool of known phytocystatins. Besides the

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study may also aid in the advancement towards the developmental strategies of transgenic plant

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variety of rai mustard (Brassica nigra), resistant to different biotic and abiotic stress conditions.

ACCEPTED MANUSCRIPT Materials and Methods Materials Cysteine proteases (Papain, ficin and bromelain), serine proteases (trypsin and chymotrypsin), substrate (casein), Sephacryl S-100 HR, electrophoresis reagents (acrylamide, bis-acrylamide, ammonium persulfate, TEMED), ethylene diammine tetra acetate (EDTA) and L-cysteine were

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purchased from Sigma (St. Louis, MO, USA). Medium range molecular weight markers,

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chemicals used were of analytical grade available commercially.

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Freund’s complete and incomplete adjuvants were procured from Genei, India Limited. All other

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Methods Isolation of cystatin from rai seeds

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Rai seed cystatin (RSC) was isolated from mustard (rai) seeds by slightly modifying the method reported earlier by Khan et al. [31]. 100 gm of prime quality rai seeds were soaked overnight at

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4ºC in homogenizing buffer (50 mM sodium phosphate buffer (pH 7.5), 3 mM EDTA, 0.15 M sodium chloride and 2% n-butanol) and subjected to centrifugation at 8,000 rpm for 15 min at

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4ºC in a sigma cooling centrifuge (3K30 model, Germany). Supernatant was collected and subjected to ammonium sulphate saturation of 40%. The suspension obtained was kept for 3 h at

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4°C and then centrifuged at 8,000 rpm for 20 min at 4°C. The supernatant thus obtained was again saturated with 40 to 60% ammonium sulphate and kept for 3 h at 4°C. Pellet retrieved after

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centrifugation of the suspension at 10000 rpm for 30 min at 4ºC was dissolved in minimum volume of 50 mM sodium phosphate buffer (pH 7.5) and dialysed thrice against hundred

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volumes of the same buffer. The sample extract obtained after dialysis was subjected to column chromatography on sephacryl S-100HR gel filtration column equilibrated with 50 mM sodium phosphate buffer (pH 7.5). Sample fractions of 5ml were collected and analysed for papain inhibitory activity and protein content. The purification process was repeated several times to obtain fresh purified inhibitor for the succeeding experiments. Cysteine protease inhibitory assay of RSC The inhibitory activity of RSC was determined by evaluating its competency to inhibit the caseinolytic activity of papain following the method reported by Kunitz [36]. Different RSC

ACCEPTED MANUSCRIPT concentrations were incubated with papain in 1 ml of 50 mM sodium phosphate buffer (pH 7.5) containing 0.047 M EDTA and 0.14 M cysteine for 30 mins at 37°C. One mL of 2% casein was further added to the mixture, followed by incubation for 30 mins at 37°C. The reaction was finally stopped by the addition of 10% TCA to the above mixture. The acid insoluble precipitates formed in the mixture were removed by centrifugation at 2500 rpm for 10 mins. Supernatant thus obtained was collected and assayed for protein concentration following the method reported by

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Lowry et al. [37]. Inhibitory activity of RSC against cysteine protease ficin, bromelain and serine

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protease trypsin and chymotrypsin was also assayed in the similar manner. Electrophoresis

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2D Gel & SDS-PAGE Electrophoresis

Two-dimensional gel electrophoresis was performed to check the homogeneity of the purified

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inhibitor. 200μg of the purified inhibitor was applied and focused on IPG strip (pI 3-10) using an IEF100 1D-Isoelectric focussing unit, Hoefer, Inc. USA. The IPG strip was further equilibrated

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with reducing and alkylating buffers by the method reported earlier by Khan et al. [38]. After equilibration, the IPG strip was transferred to SDS-polyacrylamide gel (12%) and electrophoresis

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was performed at constant voltage (100V). Staining of the gel was performed using 0.1% Coomassie brilliant blue (CBB R-250) dye. The gel was thoroughly destained using destaining

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solution (20% methanol, 10% glacial acetic acid and distilled water).

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Molecular mass determination by SDS-PAGE Molecular mass of the purified protein (RSC) was figured out by running the medium range

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protein molecular weight markers (phosphorylase b-97.4 kDa; bovine serum albumin-66 kDa; ovalbumin-43 kDa; carbonic anhydrase-29 kDa; soyabean trypsin inhibitor-20.1 kDa; lysozyme14.3 kDa) along with the purified inhibitor in the presence and absence of 2-mercaptoethanol [39] on SDS PAGE (gel concentration - 15%). Molecular mass determination by column chromatography In the native state, molecular mass was figured out by passing the purified inhibitor along with different standard protein markers on Sephacryl S-100 HR column.

ACCEPTED MANUSCRIPT Determination of Stokes radius and diffusion coeffient Stokes radius (r) of the purified inhibitor (RSC) was calculated by the method reported by Andrews [40], using standard proteins of known Stokes radii by passing them on Sephacryl S100 HR column equilibrated with 50 mM sodium phosphate buffer (pH 7.5). Data obtained was evaluated according to the equation reported by Laurent and Killander [41].

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Thiol group estimation

Thiol group of purified inhibitor (RSC) was determined by the method reported by Ellman [42]

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using dithio nitrobenzoic acid (DTNB) at a molar extinction coefficient of 13,600 M -1 cm-1.

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Standard used for the analysis was L-cysteine. Estimation of carbohydrate content

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Carbohydrate content was determined by the method reported by DuBois et al. [43] using

pH stability of the inhibitor (RSC)

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glucose as a standard.

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pH stability of RSC was checked by pre-incubating 50 μg of RSC with buffers of different pH values for 30 min at 37 °C. Buffer used were: 50 mM sodium acetate buffer (pH 3.0, 4.0, 5.0 and

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6.0), 50 mM sodium phosphate buffer (pH 7.0 and 8.0) and 50 mM Tris–HCl buffer (pH 9.0 and 10.0). Aliquots of the sample mixture were taken and assayed for the percent remaining

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RSC.

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inhibitory activity towards papain, as explained in the section of protease inhibitory assay of

Thermal stability of inhibitor (RSC) Thermal stability analysis of purified RSC was carried out by incubating RSC in 50 mM sodium phosphate buffer (pH 7.5) at different temperatures for 30 mins. Samples were rapidly cooled in ice-cold water bath and analysed for residual inhibitory activity towards papain. Immunological Assay Antibodies against the purified inhibitor (RSC) were raised in healthy male albino rabbits by subcutaneously injecting 300 μg of RSC emulsified in Freund’s complete adjuvant.

ACCEPTED MANUSCRIPT Immunodiffusion was performed by the method reported by Khaki et al. [44]. Antigen specific antibodies raised against RSC were detected by performing direct binding ELISA in the sera of RSC immunized rabbits by the method reported by Khaki et al. [44]. Active site titration and kinetics of inhibition

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Active site titration

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For the determination of binding stoichiometries, titrations between activated papain and purified RSC were carried out and the changes produced were monitored by the variations occurring in

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the fluorescence emission spectra, displaying the interactions. Method reported by Khaki et al.

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[44] was followed for the assay. Inhibition of different proteases by RSC

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Cysteine proteases (papain, ficin, bromelain) and serine proteases (trypsin and chymotrypsin), 50 μg each, were incubated with varying concentrations of RSC (0–60 μg) for 30 mins. The

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inhibitory potential of RSC towards these proteases was analysed by the method reported by

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Kunitz [36]. Casein (2%) was used as a protease substrate in the assay. Inhibition constant (ki) determination

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For the determination of inhibition constant (ki) of different cysteine proteases, varying concentrations of the inhibitor (RSC) were taken to obtain a non-linearity of dose-response

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curves. Papain, ficin and bromelain (cysteine proteases) were used at a fixed concentration of 0.20 mg/ml to react with the inhibitor in varying concentrations from 0.06 to 0.30 μM. Residual

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protease inhibitory activity of RSC was measured by the method reported by Kunitz [36], using casein as a substrate. Four different substrate concentrations, 0.5 Km, 1 Km, 2 Km and 3 Km were used for the analysis. Results obtained were analysed by Dixon [45,46] and LineweaverBurk [47] method of plotting. Determination of dissociation (k−1), association rate constant (k+1), and half-life (t1/2) value of the complexes Dissociation rate constant (k−1) was determined following the concept that maximal association takes place between protease and the inhibitor before final drift of the reaction towards

ACCEPTED MANUSCRIPT dissociation after excessive addition of the substrate. Substrate in excess binds to the entire free enzyme and leads to dissociation of the complex. Substrate prompted dissociation was witnessed with a similar protease-inhibitor complex incubated for 30 mins at 37°C. Substrate (casein) in excess (6%) was added to the reaction mixture for varying time intervals which was followed by assaying the residual activity of the inhibitor.

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Association rate constant (k+1), and half-life (t1/2) values of the complexes were obtained by

[48]

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k+1 = k-1/ki

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following relations:

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and

Spectral studies

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Ultraviolet (UV) absorption spectroscopy

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t1/2 = 0.693/k-1

UV absorption studies were performed to obtain the spectra of RSC (2.66 μM), papain and RSC-

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papain complex (molar ratio of 1:1). Absorption spectra were recorded in the range of 220 to 340 nm on Shimadzu UV Spectrophotometer (UV 1800) using a quartz cuvette of 10 mm path

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length. Absorption spectra taken were average of at least four scans.

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Fluorescence Spectroscopy

Fluorescence emission spectra of RSC, papain and RSC-papain complex (molar ratio 1:1) was

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recorded by exciting the protein samples at 280 nm on a Shimadzu RF-5301 Spectrofluorophotometer (Tokyo, Japan) for total protein fluorescence. Emission spectra were recorded in the range of 300-400 nm with a sample path length of 10 mm while keeping the slits at a width of 5 nm for both emission and excitation. RSC and papain concentrations used for the analysis were 2μM in a total volume of 1ml. Sample controls were run and necessary spectral corrections were made wherever necessary.

ACCEPTED MANUSCRIPT Circular dichroism (CD) spectroscopy CD spectra of RSC alone and RSC-papain complex (molar ratio 1:1) were obtained on a Jasco Spectropolarimeter (model J-815) in far-UV region (200-250 nm) with a quartz cell of path length 1 mm. Protein concentration for the analysis was kept at 0.2 mg/ml in 50 mM sodium phosphate buffer (pH 7.5). Procedure reported by Khaki et al. [44] was followed for the analysis

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FTIR (fourier transform infrared spectroscopy) spectral analysis

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and the calculations thereof.

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FTIR, a vibrational spectroscopy, analyses the wavelength and intensity of the absorption of infra red radiation by a given sample. It is a useful technique which aids in the determination of

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secondary structure of a protein molecule. FTIR measurements for RSC and RSC-papain complex (molar ratio 1:1) were carried out on Perkin Elmer - FTIR (Perkin Elmer Spectrum-100,

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USA) at room temperature (25°C). Spectral measurements were done in the range of 1,600-1,700

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cm-1 and each spectrum represents the average of four scans. Isothermal titration calorimetric measurements (ITC)

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To elucidate the energetics and stoichiometry of binding of RSC with papain, ITC analysis was carried out. ITC measurements were carried at 37°C on a VP-ITC titration micro calorimeter

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(Micro Cal Inc., Northampton, MA). All the sample solutions required for the analysis were filtered and degassed properly prior to the start of assay. Afterwards, reference cell, sample cell

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and syringe were loaded with 50 mM sodium phosphate buffer (pH 7.5), RSC and papain respectively. The process was followed by the subsequent injections of 10 μl of papain into the

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sample cell containing RSC. Time interval between the successive injections was 180 sec and each injection was made over 10 sec of time. Rest of the procedure was same as was followed by Khaki et al. [44].

ACCEPTED MANUSCRIPT RESULTS Purification of RSC (rai seed cystatin) Rai seed cystatin (RSC) was purified from mustard (rai) seeds by slightly modifying the method reported earlier by Khan et al. [31]. Purification process involved a simple two-step procedure comprising of ammonium sulfate fractionation and gel filtration chromatography on Sephacryl

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S-100HR column. Brief outline of the purification process is given in Table 1. Initial crude

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homogenate contained different unwanted proteins which were removed by salting out process

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using ammonium sulfate. The protein precipitate obtained after ammonium sulfate fractionation (40 – 60 %) was then dissolved in a minimum volume of 50 mM sodium phosphate buffer (pH

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7.5) and was dialysed with several volumes against the same buffer containing 0.15 M NaCl at 4°C. Purification procedure followed till dialysis resulted in 12.61-fold purification with a %

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yield of 75.55.

After dialysis, the protein sample was filtered and chromatographed on Sephacryl S-100HR

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column pre-equilibrated with 50 mM sodium phosphate buffer (pH 7.5). Protein fractions (5 ml each) were collected and assayed for protease (papain) inhibitory activity and protein content.

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Column elution profile presented a single protein peak with significant protease (papain) inhibitory activity and protein content (Figure 1). Final purification step resulted in a % yield of

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51.85 with 151.50 fold purification. Protein fractions with the peak values for papain inhibition

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were pooled for further studies.

Homogeneity of the purified RSC

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Elution profile obtained for the purified inhibitor (Figure 1) presented a single symmetric peak with constant specific activity suggesting the preparation to be homogenous in nature. Observations made were further verified by performing 2D gel electrophoresis (Figure 2). Biochemical properties of the purified RSC Electrophoretic behaviour on SDS-PAGE (Reducing and non-reducing condition) Electrophoretic behaviour of RSC was evaluated by running SDS-PAGE under reducing (SDS + βME) and non-reducing conditions (βME absent) following the method reported by Weber and

ACCEPTED MANUSCRIPT Osborn [49]. Purified inhibitor was found to migrate as a single band under both reducing and non-reducing conditions suggesting a single polypeptide structure devoid of any subunit structure (Figure 3). Molecular mass determination Molecular mass of the purified inhibitor (RSC) was resolved under native as well as under

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denaturing conditions. Under native conditions, the molecular mass was assessed by passing the

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purified inhibitor through a gel filtration (Sephacryl S-100HR) column. Marker proteins of

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standard molecular weights were also passed on the same column (after equilibration with 0.05 M sodium phosphate buffer pH 7.5) and the elution volumes were determined. Data obtained

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was analysed by the method as reported by Khaki et al. [44]. Data analysis revealed the molecular mass of the purified RSC to be 19.95 kDa and is further represented by an arrow in the

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Figure 4 (a) depicting the position of purified RSC.

Under denaturing conditions, molecular mass of the purified RSC was further established from

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the mobility in SDS-PAGE under reducing (SDS + βME) and non-reducing conditions (βME absent) following the method reported by Weber and Osborn [49]. Marker proteins were run

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simultaneously and the relative mobility (Rm) of each marker protein against the log molecular

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weight (Log M) depicted the molecular mass of the purified RSC to be 19.50 kDa (Figure 4 b). Hydrodynamic parameters

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Hydrodynamic parameters of proteins show concordance with their elution pattern from the gel filtration column. For the determination of Stokes radius, a Linear plot between Stokes radius (r)

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and [−log Kav]1/2 of the marker proteins was formulated and used for the computation of Stokes radius of RSC (Figure 5). Value for the Stokes radius of RSC was found to be 19.80 Å. In addition the value of diffusion coefficient (D) for RSC was found to be 11.21× 10-7 cm2 s-1, as calculated by the equations reported by Khaki et al. [44]. Sulfhydryl and carbohydrate group estimation Purified RSC was found to be devoid of free thiol groups or disulphide linkages. In addition, the purified inhibitor was found to lag any carbohydrate content (result not shown).

ACCEPTED MANUSCRIPT pH and thermal stability of RSC pH stability of the purified inhibitor was analysed by performing the caseinolytic activity assay for the samples kept at different pH values. From the results (Figure 6 a) it can be observed that the inhibitor is stable in the pH range of 5.0 – 9.0 and shows maximal inhibitory activity towards papain in the pH range of 7.0 - 8.0. For the pH values below 5.0 and above 10.0, the inhibitor

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showed a very low inhibitory potential. Figure 6 b represents the thermal stability profile of

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RSC kept at different temperatures. The purified inhibitor was found to be quiet stable in the

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temperature range of 30 - 60 °C and further showed a rapid loss in its inhibitory potential as the temperature was raised beyond 70 °C.

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Immunological properties

Purified RSC gave a good immune response with the resulting antiserum presenting a titre value

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of 19,952.62 as determined by direct binding ELISA (result not shown). Additionally occurrence of a single precipitin line (Figure 7) (for the antibodies raised against the purified RSC) in the

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immunodiffusion assay suggested that the wells contained apparently pure RSC depicting the

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immunogenic purity and homogeneity of the preparation.

Active site titration

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Active site titration and kinetics of inhibition

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Changes appearing in the fluorescence emission spectra for the titrations carried between activated papain and the purified RSC revealed binding stoichiometries for the interaction to be

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1:1 (Figure 8 a).

Inhibition of different proteases by RSC Purified RSC showed effective inhibition towards cysteine proteases in the order papain > ficin > bromelain while it failed to show any inhibition towards serine proteases trypsin and chymotrypsin (Figure 8 b).

ACCEPTED MANUSCRIPT Kinetics of inhibition Values of different kinetic constants for the interaction involving RSC and cysteine proteases (papain, Ficin and bromelain) were obtained by examining the loss in enzymatic activity. Purified RSC presented itself as a high affinity papain binder following the trends as reported previously for other phytocystatins [8,31]. Figure 8 c shows the Lineweaver–Burk plot in which

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the value for Vmax shows a decreasing trend with an increase in inhibition at different

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concentrations (0.06 μM – 0.3 μM) of RSC used. In addition, the values for ki (app) were found to

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remain same (Figure 8 d). This decrease in values of Vmax with similar ki (app) values on increase in inhibitor concentration clearly indicates a non-competitive mode of inhibition for RSC.

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Values for different kinetic constants ki, k+1, k-1, and t1/2 obtained on interaction of RSC with cysteine proteases (papain, ficin and bromelain) are given in Table 2. Values of kinetic constants

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obtained for ki and k-1clearly indicate that the purified inhibitor has highest affinity for papain followed by ficin and bromelain. The calculated half-life values for papain, ficin and bromelain

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were 8.07 × 102, 5.44 × 102 and 5.13 × 102 respectively (Table 2). The calculated half-life values and the values for k+1 again advocate the highest affinity of the purified RSC for papain than

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ficin and bromelain.

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Interaction of RSC with papain: Conformational analysis To analyse for the structural and conformational alterations produced in the purified inhibitor on

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interaction with cysteine protease papain; UV absorption, fluorescence emission, far-UV CD and FTIR spectroscopic studies were performed.

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UV absorption spectroscopy

RSC alone showed an absorption peak around 280 nm, typical of a protein molecule. On interaction with papain at its stoichiometric ratio, an increase in absorption intensity can be seen depicting the conformational change in either one or both of the proteins (Figure 9 a). Fluorescence emission spectroscopy Fluorescence spectroscopic studies were performed to observe for the possible conformational alterations taking place in RSC on interaction with equimolar concentration of papain. Figure 9

ACCEPTED MANUSCRIPT b shows the spectra for native RSC, native papain and RSC in complex with papain. From the figure it can be observed that native RSC shows an emission maximum around 345 nm typical for this inhibitor. On interaction with equimolar concentration of papain, an increase in fluorescence intensity along with a peak shift in maxima from 345 nm to 340 nm was observed. The observed peak shift along with increased fluorescence intensity can be attributed to the

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altered confirmation in either one or both of the proteins produced on binding.

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Far-UV CD spectral analysis

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Protein analysis by far-UV CD presents protein spectra with characteristic signature peaks of varied shape and magnitude, specific for protein secondary structural elements such as α-helix,

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β-sheet and random coil structures. Figure 9 c presents the far-UV CD spectra of native RSC and RSC in complex (equimolar) with papain. As observed in the figure, native RSC showed

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spectrum typical of a protein rich in α-helix characterized by negative peaks around 208 nm and 222 nm. The MRE (mean residual ellipticity) values for native RSC at 208 nm and 222 nm were

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found to be -11007.9 and -13034 deg cm2 dmol−1, respectively. α-helical content of native RSC was calculated from the MRE222 value and was found to be 35.29%. Method adopted for the

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calculations is same as was followed by Khaki et al. [44]. Far-UV CD analysis of RSC with papain (Figure 9 c) at its stoichiometric ratio depicts the

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conformational changes taking place in RSC upon complex formation. It can be observed from the figure (Figure 9 c) that on complex formation; there occur a significant change in the values

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proteins.

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of α-helical elliptical peaks depicting the change in conformation of either one or both of the

Fourier transform infrared spectroscopic analysis FTIR spectroscopy was aimed to analyse the secondary structural components present in the purified RSC along with the conformational changes taking place in this inhibitor upon binding with the equimolar concentrations of papain. Figure 9 d shows the FTIR spectra of native RSC and RSC in complex with papain. Native RSC was observed to show a peak around 1657 cm -1 corresponding to α-helix rich protein. On complex formation with papain, increase in spectral intensity along with an observable peak shift from 1657 cm-1 to 1652 cm-1 denotes the change in the confirmation of either one or both of the proteins. This increased band intensity with a peak

ACCEPTED MANUSCRIPT shift for the complex towards the lower wave number denotes changes taking place in the α-helix content of the purified RSC. Observations made in FTIR analysis for purified inhibitor and the complex run in parallel to the observations made in case of far-UV CD analysis. Isothermal titration calorimetric (ITC) assay ITC analysis was performed to unravel the binding energetics and binding stoichiometry for the

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interaction involving RSC and papain. Figure 10 shows the isothermogram obtained for the

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titrations performed between papain and RSC with the binding parameters derived shown in

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Table 3. Peaks presented in the upper panel of Figure 10 represent the raw data obtained on consecutive papain injections made into the sample cell holding RSC. Decrease in the peak

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intensity with each successive injection represents the heat change in μcal/s. Presence of negative heat curvation in the upper panel shows the nature of binding between RSC and papain to be

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exothermic in nature. Values obtained for enthalpy change (ΔH) and entropy change (ΔS) presented in Table 3 shows the interaction between RSC and papain to be enthalpically as well as entropically driven process with the forces involved in the interaction being electrostatic in

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nature [44]. Further positive value for ΔS and negative value for ΔG showed the binding process

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to be spontaneous in nature. Additionally the value obtained for binding stoichiometry for RSCpapain interaction by ITC analysis came out to be 0.95 ± 0.08 sites, which is well in agreement

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with the value obtained in active site titration assay.

ACCEPTED MANUSCRIPT DISCUSSION Phytocystatins comprise a group of well-characterized class of naturally occurring thiol protease inhibitors showing their functional role by controlling the activity of papain-like cysteine proteases [50]. These plant based protease inhibitors are known for their role in regulating various metabolic and other physiological processes (organogenesis, storage protein turnover,

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seed germination) in plants. In addition, the role of phytocystatins has also been seen in plants

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under different biotic and abiotic stress conditions [28–30,50]. With the myriad of such roles

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played by these plant based protease inhibitors, it has become imperative to explore similar such protease inhibitors from novel sources in order to delineate the structural and functional capability with regard to the target proteases and also the need for improvement thereof under

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the current challenging environmental conditions. Taking all this into consideration, present study focussed on the isolation, purification and characterization of a cystatin like thiol protease

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inhibitor from the seeds of a novel source i.e. Brassica nigra (rai mustard), a commonly used

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spice cultivated worldwide with medicinal [32–35] and oil yielding properties. The isolation and purification process involved a simple two step method of ammonium sulphate

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precipitation (40 – 60 %) and gel filtration chromatography (Sephacryl S-100HR) leading to a final yield of 51.85 % with a fold purification of 151.50 from 100 gm of rai mustard seeds

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(Table 1). The procedure followed is simple and very efficient as it gave a better yield than the other reported procedures [31,44,51,52]. Purity and homogeneity of the isolated RSC was

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ascertained by the emergence of a single symmetric peak in the elution profile with significant papain inhibitory activity (Figure 1) and was verified from 2D-gel electrophoresis (Figure 2).

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Purified inhibitor migrated as a single band on SDS-PAGE under reducing and non-reducing conditions suggestive of a single polypeptide chain devoid of any subunit structure and disulphide bonds (Figure 3). Purified RSC presented a molecular mass of 19.95 kDa on passing through a gel filtration column (Sephacryl S-100HR) under native conditions Figure 4 (a). Under denaturing conditions, the molecular mass was confirmed to be 19.50 kDa (Figure 4 b). Phytocystatins with molecular mass of 26 kDa, 23 kDa, 18.1 kDa, 12.5 kDa have been reported from yellow mustard seeds (Brassica alba) [8], Brassica rapa flower buds [53], Brassica juncea seeds [31] and garlic phytocystatin (GPC) [54] respectively. Cystatins type I and type II in general have been categorised on the basis of molecular mass, molecular complexity, biological

ACCEPTED MANUSCRIPT distribution, presence or absence of disulphide bonds and carbohydrate content [1,44], while plant cystatins are sometimes considered as the intermediate form between the type 1 and type 2 cystatins. Purified RSC was found to be devoid of thiol groups and carbohydrate content. Observations made for the purified RSC are in concordance with the properties that identify a protein as a group II phytocystatin [2]. Hydrodynamic properties were worked out to provide an insight about the size and shape of the purified RSC [55]. Stokes radius of the purified inhibitor

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as figured out from its behaviour on the gel filtration column was found out to be 19.80 Å

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(Figure 5). Value of stokes radius for RSC (19.80 Å) was quiet comparable to the lysozyme

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(18.60 Å) and trypsin (20.20 Å), thus suggesting that RSC is a protein with globular shape. Similar such values for the stokes radius have also been reported for cystatin types purified from

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yellow mustard seeds (Brassica alba) [8] (23 Å) and the seeds of Brassica juncea (20.70 Å) [31]. Likewise the value of diffusion coefficient (D) for the RSC was found to be 11.21× 10-7

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cm2 s-1 and was quiet comparable to the values obtained for yellow mustard seeds (Brassica alba) [8].

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To mark the stability profile of RSC, an attempt was made to assess its papain inhibitory potential at different pH and temperature levels for varying time intervals. It was observed that

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the purified inhibitor was quiet stable in the pH range of 5.0 – 9.0 and shows maximal inhibitory activity towards papain in the pH range of 7.0 - 8.0 (Figure 6 a). Reports suggest that majority

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of the plant based cystatins show stability towards the extremes of pH and temperature [8,31,56,57]. In a similar manner, behaviour of purified RSC in the temperature range of 30 - 60

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°C holds good (Figure 6 b). Antibodies against the purified RSC were raised with the build-up of titre signifying an extent to which an organism produces antibodies by recognizing a particular

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epitope, which further is expressed as the inverse of the greatest dilution, presenting a positive result. Purified RSC evoked a fair immune response with an antibody titre of 19,952.62 as determined by direct binding ELISA. Similar such values of 22,000 and 23442.29 have also been reported for the purified plant cystatin forms from the seeds of yellow mustard (Brassica alba) with a molecular mass of 26 kDa [8] and from the seeds of Brassica juncea [31] with a molecular mass of around 18.10 kDa respectively. Additionally, antibodies raised presented a reaction of individuality with the purified inhibitor as revealed by a single precipitin line on immunodiffusion, suggesting that the wells contained apparently pure RSC. Further results also denoted the immunogenic homogeneity of the purified inhibitor (Figure 7).

ACCEPTED MANUSCRIPT Stoichiometric and kinetic profiling of the purified inhibitor with different proteases was done to unravel the number of binding sites and the values thereof for different kinetic parameters. Binding stoichiometry between papain and the purified inhibitor was found to be 1:1 (Figure 8 a), signifying that one papain binding site is present per RSC molecule. Similar such stoichiometric ratios have also been reported for different plant [31] and animal cystatin types [44,51,58,59]. Stoichiometric ratios other than 1:1 have also been reported with inhibitor-papain

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binding ratio of 1:1.1 observed for Clitocypin [60] and 1:8 for a cystatin type isolated from the

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leaves of methyl jasmonate treated tomato plants [11]. Pure and homogenous fractions obtained

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for the inhibitor as such were found to be highly specific for cysteine proteases (papain, ficin and bromelain) while they showed no inhibition towards serine proteases (trypsin and chymotrypsin)

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(Figure 8 b). Among the cysteine proteases, RSC was found to be maximally effective towards papain followed by ficin and bromelain. Values obtained for different kinetic parameters on

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kinetic profiling of RSC with different proteases revealed that the inhibitor shows highest affinity towards papain with ki of 1.62 × 10-7 M and an association rate constant (k+1) of 5.30 × 103 (M-1 s-1) followed by the values obtained for ficin and bromelain (Table 2). The ki values

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obtained for papain are quiet comparable to the ones reported for Brassica juncea (ki: 1.02 × 10-7

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M) [31], yellow mustard seeds (Brassica alba) (ki: 3 × 10-7 M ) [8] and the dimeric form of Chinese cabbage (ki: 1.01 × 10-7 M ) [57]. Earlier reported ki values for strawberry [61], barley

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[62] , oryzacystatin-I [63] and sesame [64] were found to be of higher order in magnitude. Further among the mustard cystatin forms, RSC was found to be more potent inhibitor than

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yellow mustard (Brassica alba) cystatin form [8]. Additionally, ki values obtained for the purified RSC also go well with the corresponding values obtained for mammalian cystatin types

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[65,66]. Tight binding inhibitors are known for their high association rate constants values and in the present study association rate constant values followed the order papain > ficin > bromelain (Table 2) signifying that RSC has high affinity towards papain and lower affinity towards bromelain. Further weaker biding of the purified inhibitor towards bromelain can largely be attributed to a low dissociation rate constant, k-1 (Table 2). Half-life (t1/2) values calculated for the protease – inhibitor complex for papain, ficin and bromelain were found to be 8.07 × 102, 5.44 × 102 and 5.13 × 102 respectively (Table 2) indicating papain – RSC complex to be more stable than either of the ficin or bromelain complexes. Similar trend for the half-life (t1/2) values have been reported for yellow mustard (Brassica alba) cystatin [8], cystatin from Brassica

ACCEPTED MANUSCRIPT juncea [31], garlic (Allium sativum) phytocystatin (GPC) [54], cystatin from caprine brain (CBC) [44] and goat pancreatic cystatin [51]. The inhibition constants presented in Table 2 suggests that RSC follows a non-competitive mode of inhibition which was also visualised from Lineweaver–Burk (Figure 8 c) and Dixon plot (Figure 8 d). Non-competitive pattern of inhibition has also been previously been reported for strawberry [61] and soyabean cystatin [67].

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Purified inhibitor was also explored for structural and conformational properties alone and/or in

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association with cysteine protease; papain by employing UV absorption, fluorescence emission,

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far-UV CD and FTIR spectroscopy. RSC alone showed an absorption peak around 280 nm, typical of a protein molecule while on interaction with equimolar concentration of papain, conformational change takes place, possibly leading to exposure of absorbing groups to polar

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environment thereby causing perturbations around aromatic residues eventually leading to intensity change and red shift (Figure 9 a). The findings are quiet comparable to the earlier

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reported results [54,68,69]. Further an emission maximum around 345 nm for native RSC presented the signature fluorescence peak for this protease inhibitor which on interaction with

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equimolar concentration of papain shows enhancement in intensity with a peak shift representing the structural distortion (Figure 9 b). The observable intensity change and peak shift was

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prominent between 335 – 345 nm indicating the changes arising largely from perturbations around tryptophan and/or other aromatic amino acid residues. Secondary structural analysis by

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far-UV CD revealed α-helical content for the purified inhibitor to be 35.29 % (Figure 9 c). A variety of reports are available documenting similar such α-helical values for both plant [54,69]

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and animal cystatin [44,68] types thus presenting the homologous nature of the purified inhibitor with such cystatin forms. Further figure 9 c represents an observable α-helical peak shift for the

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native RSC form when in complex with papain depicting the structural alteration. A good number of reports are available presenting the change and/or shift in α-helical peaks for the cystatin–papain interaction [8,44,54,65,66,69]. FTIR spectral analysis of native RSC and RSC in complex with papain was further performed to gather information about the secondary structural elements and the deviations in the structure if any upon complex formation. Spectra of the native RSC gave peak around 1657 cm-1, which corresponds to a protein rich in α-helix (Figure 9 d). Further upon complex formation with

ACCEPTED MANUSCRIPT equimolar papain concentration a peak shift with increased intensity was observed (Figure 9 d), further confirming the observations made in far-UV CD analysis. Biological processes are considered to be complete and effective only when there is highly specific complex formation between the proteins involved. A number of driving forces are involved which directly contribute to the concerned interactions in such specific biological

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complexes. Forces at display may involve hydrogen bonding, van der Waals forces, electrostatic

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forces, hydrophobic interactions etc. To understand and characterize such driving forces and the

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interactions involved, ITC analysis was performed. Values obtained for different thermodynamic parameters are presented in Table 3, suggesting the process to be enthalpically as well as entropically driven with the nature of binding between the proteins to be exothermic. Negative

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enthalpy (ΔH) and positive entropy (ΔS) values suggested the interaction forces involved to be electrostatic in nature [44,70]. Further the values obtained presented the incidence of

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conformational changes in the proteins involved in complexation [44,71], thereby confirming the results obtained for spectroscopic analysis. Results obtained in ITC analysis were also in full

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agreement with those obtained for kinetic parameters.

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Summing it all, the present study gives an insight about different characteristic properties of a cystatin like thiol protease inhibitor from the seeds of rai mustard (Brassica nigra). The study

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will not only aid in determining the functional behaviour of the purified inhibitor during various physiological and other stress conditions in the rai mustard but might also help in improving the

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Conclusion

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crop variety thereby improving the crop yield, hence meeting the increased demands.

Recent advances in phytocystatin research concerning their characterization and function has shown quality potential in deciphering their role in various physiological processes thereby showing their involvement in the regulation of various endogenous or exogenous proteases. Role of phytocystatins in plant defence mechanism has pushed researchers to consider these protease inhibitors as proteins of tremendous value with potential based transgenic expression thereby proving to be new tools in pest control management and giving plants the ability to overcome different biotic and abiotic stress conditions, hence increasing the gross productivity and crop yield. Concurrently, present study shows the structural and functional characteristics of RSC

ACCEPTED MANUSCRIPT with resemblance to the already known phytocystatin types, thence adding up to that pool. With the advantage of being small in size and being able to withstand a wide range of temperature and pH, RSC gets an advantage to be used as a putative target for genetic modification so as to be used potentially in medicine, agriculture, and food technology.

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ACKNOWLEDGMENT

The work was supported by the financial assistance provided under the DBT-BUILDER program

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(No.BT/PR4872/INF/22/150/2012). Facilities provided by the Department of Biochemistry,

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Aligarh Muslim University, Aligarh are highly acknowledged.

The University Grants Commission (UGC), New Delhi (India) is gratefully acknowledged for

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the award of BSR Faculty Fellow to Prof. Bilqees Bano, Dept. of Biochemistry, F/O: Life Sciences, AMU, Aligarh (India)

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Facilities provided by Integral University, Lucknow are highly acknowledged. This manuscript

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has communication no. IU /R&D/2018- MCN000471

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CONFLICT OF INTEREST: The authors declare that there is no conflict of interest in this

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work.

ACCEPTED MANUSCRIPT FIGURE LEGENDS

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Figure 1. Gel filtration contours of dialysed RSC: Protein precipitate obtained after 40 to 60% ammonium sulphate fractionation was loaded on Sephacryl S-100HR gel filtration column. Fractions of 5 ml were collected and assayed for the inhibition of caseinolytic activity of papain and absorbance at 660 nm. Fractions with maximum inhibitory activity (12, 13 and 14) were pooled for further studies.

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Figure 2. 2D gel electrophoresis of the purified RSC: 200μg of the purified inhibitor was applied and focussed on an IPG strip followed by subjecting to SDS-polyacrylamide gel (12%) electrophoresis. A single spot observed on the gel suggested the purity and homogeneity of the sample.

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Figure 3. SDS-PAGE of RSC under reducing and non-reducing conditions: SDS-PAGE of RSC was carried out on 15% polyacrylamide gel. Middle lane contained the medium range molecular weight marker proteins: A, Phosphorylase b (97.4 kDa); B, Bovine Serum Albumin (66 kDa); C, Ovalbumin (43 kDa); D, Carbonic Anhydrase (29 kDa); E, Soyabean Trypsin Inhibitor (20.1 kDa); F, Lysozyme (14.3 kDa). Lane a contained RSC without βmercaptoethanol and lane b contained RSC along with β-mercaptoethanol.

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Figure 4(a). Molecular mass determination by gel filtration chromatography: Plot of log M vs Ve/Vo for the estimation of molecular mass of the purified RSC using Sephacryl S-100HR column. Marker proteins passed through the column are: A- Cytochrome C (12.3 kDa); BLysozyme (14.3 kDa); C- Trypsin (23.3 kDa); D- Chymotrypsin (25 kDa); and E- BSA (66 kDa). Arrow shows the position of RSC.

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Figure 4(b). Molecular mass determination by SDS-PAGE under reducing and nonreducing conditions: Log M of marker proteins was plotted against their relative mobility (Rm) for determination of the molecular mass of purified RSC. Molecular weights of standard marker proteins are given in material & methods section. Molecular mass of the purified RSC was found to be 19.50 kDa and the position is represented by an arrow.

Figure 5. Stokes radius (r) determination of the purified RSC by Laurent and Killander plot [-(log Kav)1/2 vs stoke’s radius (r)]: Marker proteins and the purified RSC were subjected to gel filtration on Sephacryl S-100 HR column. Stokes radii of the marker proteins were: ABovine Serum Albumin (35.50 Å); B- Ovalbumin (27.30 Å); C- Trypsin (20.20 Å), D-

ACCEPTED MANUSCRIPT Lysozyme (18.60 Å), E- Cytochrome C (17.50 Å). Arrow shows stokes radius (19.80 Å) for the purified RSC.

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Figure 6(a). pH stability profile of purified RSC: 50 µg of the purified RSC was incubated in 50 mM sodium acetate buffer (pH 3.0–6.0), 50 mM sodium phosphate buffer (pH 7.0–8.0) and 50 mM Tris-HCl buffer (pH 9.0-10.0) for 30 min at 37 oC. Remaining % inhibitory activity was analyzed against papain at 37 oC.

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Figure 6(b). Effect of temperature on the purified RSC: Purified RSC was incubated in 50 mM sodium phosphate buffer (pH 7.5) at various temperatures for 30 min. Remaining % inhibitory activity was analyzed against papain at 37 oC.

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Figure 7. Ouchterlony immunodiffusion: For the immunodiffusion study, anti-RSC antiserum was raised in male albino rabbits. The antiserum was allowed to react with RSC on agarose plates. Central well contained the antiserum, whereas the surrounding three wells (A, B and C) contained the purified RSC.

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Figure 8(a). Inhibition of papain by RSC as monitored by intrinsic fluorescence: Excitation wavelength used was 280 nm while emission wavelength was 350 nm. Fp: fluorescence of protease. Fc: fluorescence of added RSC. Fp.c: Fluorescence of protease/RSC mixture.

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Figure 8(b). Inhibitory activity of RSC with different proteases: 50 μg of thiol proteases (papain, ficin, bromelain) and serine proteases (trypsin and chymotrypsin) were incubated with varying concentration of RSC (0-60μg) for 30 min. The inhibitory activity of RSC towards different proteases was measured using casein as substrate.

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Figure 8(c). Lineweaver–Burk plot showing inhibition of papain by purified RSC: Plot represents the inhibitory effect of the purified inhibitor in the presence of its different concentration values ranging from 0.06 μM to 0.30 μM on papain. Figure 8(d). Dixon plot showing the apparent ki [ki (app)] value for RSC: Plot gives the apparent ki by plotting 1/Vmax vs [I] after obtaining Vmax from Lineweaver–Burk plot.

Figure 9(a). UV absorption spectra of RSC, Papain and RSC-Papain complex: Absorption spectra of RSC, Papain and RSC-Papain complex were recorded in the range of 220-340 nm. RSC and papain were in the molar ratio of 1:1.

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Figure 9(b). Fluorescence spectra of RSC, papain and RSC-papain complex: Fluorescence spectra were taken at an excitation wavelength of 280 nm and recording the emission between 300-400 nm. Papain and RSC was used at a molar ratio of 1:1.

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Figure 9(c). Far-UV CD spectra of native RSC and RSC-papain complex: Concentration of RSC for far-UV CD analysis was 0.2 mg/ml and RSC-papain was in the molar ratio of 1:1. Buffer used was 50 mM sodium phosphate buffer (pH 7.5). Cells of 1 mm path length were used for the measurements. Observed ellipticity was presented as MRE.

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Figure 9(d). FTIR spectra of native RSC and RSC-papain complex: RSC and papain were analyzed at a molar ratio of 1:1. Spectral measurements were done in the range of 1,600-1,700 cm-1 and each spectrum represents the average of four scans.

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Figure 10. Isothermal titration calorimetric analysis for RSC titrated with papain: Upper panel represents the isothermogram and lower panel represents the binding isotherm generated after the successive injections of papain into RSC.

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ACCEPTED MANUSCRIPT Table 1. Rai seed cystatin during various stages of purification

Specific activity (units/m g) 0.013

9900

0.45

135

20

31

620

5.10

15

2.36

35.40

4.66

% Yiel d

1

100

102

0.164

CR

12.61

75.5 5

US

IP

33

Fold Purificatio n

T

Total activit y units⃰

300

M

Crude homogenat e 40-60% ammonium sulphate precipitatio n Gel filtration S-100HR

Volum Protei Total Activity e n Protei units/ml/mi (ml) mg/ml n (mg) n

70

1.97

151.50

51.8 5

AN

Step of purificatio n

AC

CE

PT

ED

⃰ One unit of enzyme inhibitory activity is defined as the amount of inhibitor bringing about 0.001 change in O.D./ml/min

ACCEPTED MANUSCRIPT

ki (M)

k+1 (M-1 s-1)

k-1 (s-1)

t1/2 (s)

Papain

1.62 × 10-7

5.30 × 103

8.58 × 10-4

8.07 × 102

Ficin

3.86 × 10-7

3.30 × 103

12.73 × 10-4

5.44 × 102

Bromelain

4.82 × 10-7

2.80 × 103

13.49 × 10-4

5.13 × 102

US

CR

T

Protease

IP

Table 2. Values of kinetic constants obtained on the interaction of RSC with proteases

AC

CE

PT

ED

M

AN

Results represent the mean ± SEM calculated from three independent experiments

ACCEPTED MANUSCRIPT

Table 3. Binding and thermodynamic parameters obtained on titration of papain with RSC by ITC experiment, at 37 °C.

Parameters

Values

1

N (binding stoichiometry, RSC:Papain)

2

ΔH (binding enthalpy, kcal mol-1)

3

ΔS (change in entropy, cal mol−1 deg−1)

4

TΔS (kcal mol-1)

5

ΔG (Gibbs free energy change, kcal mol-1)

0.95 ± 0.08 sites

CR

-1.75 ± 0.45

US

11.92

AN

M ED PT CE AC

IP

T

S. No.

3.69 -5.44

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10