Inhibited Papain Activity Research Articles

Ratiometric Fluorescent Quantum Dot-Based Biosensor for Chlorothalonil Detection via an Inner-Filter Effect.

A new sensitive sensor for detecting chlorothalonil (CHL) based on the inner-filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent quantum dots (RF-QDs) was developed. Here, RF-QDs were designed by two different color CdTe QDs. Based on the IFE, the AuNPs can quench the fluorescence of the RF-QDs. Because of the electrostatic attraction between protamine (PRO) and the AuNPs, the PRO can restore fluorescence effectively. Papain (PAP) can easily hydrolyze PRO and causes the quench of fluorescence quenching. The addition of CHL can inhibit PAP activity and restore the fluorescent signal. Through the characterization of the structural changes of PAP, the inhibition and mechanism of CHL on PAP activity were studied. The ability of CHL to inhibit PAP activity was evaluated by measuring the fluorescence of the RF-QDs. Under the optimal conditions, this sensing platform shows a response to CHL in the range of 0.34-2320 ng/mL and a detection limit of 0.0017 ng/mL. Based on the CHL inhibition of PAP activity, the RF-QDs showed good selectivity for CHL. The practical application of the proposed system was demonstrated by detecting CHL in food and environmental samples with satisfying results.

Characterization of Siam Tulip (Curcuma alismatifolia) Cystatin (CaCPI)

Cystatin is a cysteine protease inhibitor that can regulate the proteolytic process of cysteine proteases by binding to the active site of those target enzymes. Plant cystatin has several reported roles, including in: protein turnover during development; programmed cell death; and plant defense mechanisms against insects, nematodes and phytopathogenic fungi. In this experiment, the CaCPI gene was isolated from the cDNA library of the Siam tulip (Curcuma alismatifolia cv. Chiang Mai Pink). Its full length cDNA sequence is 601 base pair, containing 372 base pair in an open reading frame encoding 123 amino acids and 32 and 197 base pair in the 5’and 3’ untranslated regions, respectively. The deduced amino acid sequence consists of a putative N-terminal secretory signal peptide of 22 amino acids and an estimated molecular mass for the mature protein of 11.239 kDa. The CaCPI protein contains all of the highly conserved blocks, including Gly 31 -Gly 32 , the reactive site motif QXVXG (Q 76 V 77 V 78 A 79 G 80 ), P 106 -W 107 , and the LGRFAVDQHN block that are common among plant cystatins. The CaCPI gene was cloned into a pDEST17 expression vector and then transformed into the Escherichia coli strain BL21-Star for recombinant CaCPI protein production. After induction with 1 mM IPTG, the cell lysate of E. coli carrying pDEST17-CaCPI generated a CaCPI protein about 12 Kda in size on SDS-PAGE. This cell lysate inhibited papain activity.

Expression of barley leaf cysteine proteinase inhibitor as an active fusion protein inEscherichia colicells

Nowadays, identification of the novel physio-biological and therapeutic functions of plant cysteine proteinase inhibitors “plant cystatins / phytocystatins” are the great of interests for molecular biologists. Whether for biochemical, structural or functional studies, their successful expression along with an easy purification method is required. To date, fusion tags are the best available tools that meet all those requirements. We report here the cloning and simple functional expression and purification of a barley putative cystatin in Escherichia coli cells. For the first time, a part of barley coding sequence containing a predicted active cystatin was amplified by polymerase chain reaction and expressed as maltose binding fusion protein in TB1 strain of E. coli cells using pMALc2X over-expression vector system without affecting the bacterial growth. The expressed product was purified by single step affinity chromatography from the soluble fraction of induced culture at a yield of about 37 mg/ liter of bacterial cell culture. The purified fused protein could efficiently inhibit papain activity in vitro without the cleavage of the fusion partner.

The Effect of Various Wound Dressings on the Activity of Debriding Enzymes

To understand the compatibility between the debriding enzymes collagenase and papain, and various wound dressings. The extracts from a silver dressing (Acticoat; Smith & Nephew, St Petersburg, Florida), iodine dressings (Iodoflex and Iodosorb; Smith & Nephew), a pigment-complexed polyvinyl alcohol (PVA) dressing (Hydrofera Blue; Healthpoint, Ltd, Fort Worth, Texas), and collagen dressings (Hydrofera Blue and FibraCol Plus; Systagenix Wound Management, Quincy, Massachusetts) were examined in vitro with collagenase and papain (papain was used in papain-urea debriding agents, no longer available on today's US market). All testing was in vitro and performed at Healthpoint, Ltd. Testing was not performed using human or animal subjects. All in vitro testing was conducted in the lab using artificial wound eschar substrate and other lab equipment. The main outcome measure was percent collagenase and papain activity lost when combined with each type of dressing tested. The results demonstrated that the pigment-complexed polyvinyl alcohol dressing and the collagen dressing were compatible with collagenase, whereas the iodine dressings inhibit the activity of collagenase. The nanocrystal silver dressing (Acticoat) caused more than a 50% loss in activity when combined with collagenase. Papain displayed varying levels of inhibition with all dressings tested with the enzyme. The iodine dressings significantly inhibit papain activity, whereas the other dressings exhibited inhibitory activity ranging from 10% to 30%. Antimicrobial dressings are widely used for management of wound bioburden. Frequently, they are used in combination with other topical therapeutic drugs, such as enzymatic debriding agents for the removal of wound necrotic tissues. Such combined applications may have greater potential to achieve multiple healing activities simultaneously, including exudate and bioburden management, debridement, and tissue regeneration. Overall, the authors' testing found that collagenase was observed to be more tolerant when used with the dressings tested than papain. These findings merit further exploration in clinical wounds to confirm clinical validity.

Two Kunitz-Type Inhibitors with Activity Against Trypsin and Papain from Pithecellobium dumosum Seeds: Purification, Characterization, and Activity Towards Pest Insect Digestive Enzyme

Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.

Light-induced inhibition of papain by a {Mn–NO}6 nitrosyl: Identification of papain–SNO adduct by mass spectrometry

Modification of Cys25 at the active site of the cysteine protease papain by S-nitrosylation inhibits its hydrolytic ability. Previous studies have demonstrated that NO donors N-nitrosoanilines inhibit papain activity via formation of S–NO bond formation at the active site while NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP), N-nitrosoaniline derivatives, and S-nitroso-glutathione (GSNO) inhibit the enzyme via S-thiolation by thiyl radicals generated from the S-nitrosothiols. In this study, we report papain inactivation by a photosensitive {Mn–NO}6 nitrosyl [(PaPy3)Mn(NO)](ClO4) (1) where PaPy3- is the anion of the designed ligand N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide. This nitrosyl releases NO upon exposure to visible light of low intensity (50W tungsten lamp). With Nα-benzoyl-l-arginine-p-nitroanilide (l-BApNA) as the substrate, the dissociation constant for the breakdown of the enzyme–inactivator complex (KI) and the overall inactivation rate constant (ki) were calculated to be 2.46mM and 64.8min−1, respectively. The papainS–NO adduct has been identified using electrospray mass spectrometry (ESI-MS). The results demonstrate that controlled inactivation of papain can be achieved with the {Mn–NO}6 nitrosyl 1 and light. The reaction is clean and the extent of inactivation is directly proportional to the exposure time.

Inhibition of Mammalian Legumain by Some Cystatins Is Due to a Novel Second Reactive Site

We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The Ki value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with Ki values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-Asn-NHPhNO2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low Mr kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (Ki 0.0016 nM) and relatively weak (Ki 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the alpha-helix and the first strand of the main beta-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (Ki 1.5 nM) but >>5,000-fold lower affinity for legumain (Ki >>1,000 nM) than wild-type cystatin C.

The Salivary Lipocalin Von Ebner's Gland Protein Is a Cysteine Proteinase Inhibitor

The lipocalins make up a heterogeneous superfamily of proteins. Although showing almost no sequence homology, they share very similar secondary and tertiary structures. Their ability to bind hydrophobic ligands is well established, but the physiological function of most lipocalins remains unclear. The lipocalin from the human Von Ebner's Gland of the tongue (VEGh) contains three sequence motifs corresponding with the papain-binding domains of cystatins, a family of naturally occurring cysteine proteinase inhibitors. We found that VEGh inhibited papain activity to a similar extent as salivary cystatin S. Furthermore, synthetic peptides derived from VEGh and cystatin C, comprising these three motifs, inhibited papain, too. We conclude that VEGh is a physiological inhibitor of cysteine proteinases and therefore can play a role in the control of inflammatory processes in oral and ocular tissues.

Amino Acids and Peptides. XLIV. Synthesis of Stereoisomeric Pentapeptides of Thiol Proteinase Inhibitor.

Stereoisomers of thiol proteinase inhibitor (TPI) were synthesized by a conventional solution method. Among them, iNoc-D-Gln-Val-Val-Ala-Ala-pNA weakly inhibited the amidolytic activity of papain, although iNoc-Gln-Val-Val-Ala-Ala-pNA inhibited papain activity fairly potently. However, the other five D-amino acid-containing peptides did not show any inhibitory effects on papain. The circular dichroism (CD) spectra of the enzyme-peptides mixtures were measured in order to study the relationship between the peptide anilides-papain interaction and the inhibitory activity.

パッションフルーツ中のプロテアーゼインヒビター

A protease inhibitor (PEPI) was isolated and purified from the juice of the passion fruit (Passiflora edulis Sim) by DEAE-Sephacel and trypsin-Sepharose 4B column chromatography. The molecular weight of PEPI was found to be 25 kDa by gel filtration. PEPI exhibited a single band in native and SDSPAGE gels under non-reducing conditions. Under reducing conditions in SDS-PAGE gel, PEPI exhibited three protein bands with estimated molecular weights of 25, 22, 18 kDa, respectively. The three protein bands were stained with PAS reagent. PEPI inhibited trypsin and chymotrypsin activities, but did not inhibit papain activity. The trypsin-inhibitory activity of PEPI was heat-stable, retaining 50% of the original activity even after exposure to 100°C for 10 min. The trypsin-inhibitory activity of PEPI was stable over a pH range of 2-12.

Studies on the mechanism of action of colloidal bismuth subcitrate. I. Interaction with sulfhydryls.

The present study was designed to examine the reaction pathway of colloidal bismuth subcitrate (CBS) with thiols. Studies were performed using the monothiol glutathione (GSH), the dithiol dithiothreitol (DTT) and the thiol enzymes papain and H+/K(+)-ATPase. UV-vis spectra showed that CBS forms complexes with GSH and DTT. The GSH/CBS complex but not the DTT/CBS complex was cleared by 5,5'-dithiobis-(2-nitrobenzoic acid). CBS inhibited H+/K(+)-ATPase (IC50: 23 +/- 6.5 mumol/l) but failed to inhibit papain activity. The inhibitory action of CBS on H+/K(+)-ATPase-mediated proton transport was prevented by the dithiol dithioerythritol but not by GSH. These results indicate that CBS forms stable complexes with dithiols and instable complexes with monothiols. We suggest that some of the effects of CBS (i.e., stimulation of prostaglandin production, antibacterial action against Helicobacter pylori) are mediated via the blockade of SH-groups.