Retaining in-gel zymographic activity of cysteine proteases via a cysteine-supplemented running buffer.

Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins.

Characterization of alpha-2-macroglobulin from groupers.

Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This study conducted the immuno-analysis and mass spectrometric analysis methods to investigate the characteristics of the protease inhibitor, α-2-M, among groupers and related species. Rabbit antiserum to the purified α-2-M of Epinephelus coioides was used in different immunological methods to determine the immune cross-reactions of the α-2-M in samples. Plasma of Epinephelus bruneus, Epinephelus fuscoguttatus, Epinephelus lanceolatus, and Epinephelus quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. To purify the α-2-M protein, plasma protein of grouper E. coioides was first precipitated by using PEG 6000, then Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Separose 4B and Phenyl Sepharose High Performance columns were used on FPLC system for purification. The molecular mass of grouper plasma α-2-M was determined as a 180 kDa protein on non-reduced SDS-PAGE. In addition, it was determined as 97 and 80 kDa protein on reduced SDS-PAGE. Enzymatic and chemical deglycosylation of glycogen revealed that the contents of glycogen in 97 and 80 kDa subunits were 12.4% and 15%, respectively, and were all belonging to N-linked type. Only one precipitation arc was visualized in all plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed stronger responses than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80, 97, 160, 250 kDa) were detected on reduced SDS-PAGE when various grouper plasma was performed respectivity. However, no band was detected using plasma from the freshwater fish species and other animal species. Thus, further indicates that the protein structure of α-2-M of Epinephelus spp. was closely related among seawater fish species. In addition the identity of the two subunits was identified using LC/MS/MS which was similar to α-2-M of grass carp (Ctenopharyngodon idella) and bluegill sunfish (Lepomis macrochirus) on the protein hit.

Improvement of the enzyme performance of trypsin via adsorption in mesoporous silica SBA-15: hydrolysis of BAPNA.

The enzymatic performance of trypsin in hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide (BAPNA) was improved by adsorption on Santa Barbara Amorphous (SBA)-15 mesoporous silica. The optimal immobilization conditions were screened and the properties of immobilized enzyme have also been studied. Under the optimal conditions, the immobilized trypsin displays maximum specific activity (49.8 µmol/min/g). The results also indicate that the immobilized trypsin exhibits better storage stability.

Analyzing Blood Digestion: Estimation of Active Trypsin Levels in the Mosquito Midgut.

The Nα-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) assay is widely used to quantify trypsin in mosquito midguts and is highly sensitive. BApNA is a chromogenic substrate for proteolytic enzymes such as trypsin and amidase. Hydrolysis of BApNA at the bond between the arginine and the p-nitroaniline moieties releases the chromophore p-nitroaniline, which is detected by colorimetric analysis. The intensity of the color is directly proportional to the amount of trypsin in the solution. Here, we present a trypsin measurement assay specifically using the BApNA substrate.

A green and bio-inspired process to afford durable anti-biofilm properties to stainless steel.

A bio-inspired durable anti-biofilm coating was developed for industrial stainless steel (SS) surfaces. Two polymers inspired from the adhesive and cross-linking properties of mussels were designed and assembled from aqueous solutions onto SS surfaces to afford durable coatings. Trypsin, a commercially available broad spectrum serine protease, was grafted as the final active layer of the coating. Its proteolytic activity after long immersion periods was demonstrated against several substrata, viz. a synthetic molecule, N-α-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA), a protein, FTC-casein, and Gram-positive biofilm forming bacterium Staphylococcus epidermidis.

In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti.

BACKGROUND: The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes. RESULTS: We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k(cat)/K(M)) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin. CONCLUSIONS: These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

Nafamostat mesilate, a potent tryptase inhibitor, modulates periodontitis in rats.

Previous reports have demonstrated increased tryptase-like proteolytic activity in the crevicular fluid of patients with periodontal disease. In the present study, we have investigated the effect of tryptase inhibition with nafamostat mesilate (NM, 6-amino-2-naphtlyl p-guanidinobenzoate dimethansulfonate) on the development of experimental periodontitis in rats. Eighty (80) male Wistar rats were randomly separated into four groups: Control group, NM group (daily 0.1 mg/kg body weight of NM, i.p.), Ligature group (ligature placed at lower right first molars), and NM+Ligature group. The amount of alveolar bone loss (ABL) around the mesial root surface of the first mandibulary molar, as well as the myeloperoxidase (MPO) activity, and total proteolytic activity [N-benzoyl-L: -arginine-p-nitroanilide (BApNA) substrate] were determined at 7 and 14 days. NM led to significantly (p < 0.05) decreased ABL in animals subjected to ligature-induced periodontitis. Tryptase inhibition prevented the onset of significant ABL at 7 days of experiment (0.44 ± 0.16 and 0.60 ± 0.22, p > 0.05, NM+Ligature and Control, respectively) and significantly decreased the ABL at 14 days (0.97 ± 0.17 versus 1.82 ± 0.26, p < 0.001, NM+Ligature versus Ligature, respectively). In addition, NM significantly decreased MPO and total proteolytic activity at 14 days (p < 0.05). These data provided evidence that tryptase inhibition with NM attenuates gingival granulocyte infiltration and ABL in an experimental model of periodontitis in rats.

Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest, Eurygaster integriceps.

In the current study the effects of serine proteinase inhibitors (TLCK, TPCK, SBTI, and a combination of SBTI and TPCK) with concentrations of 1% and 4% of dietary protein in artificial diets were tested against growth of the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), development, and its gut serine proteinase targets. Analysis of variance indicated that protease inhibitors affected nymphal development time, adult weight, and survival. Mean development time of third instar nymphs in control, SBTI (1%), TLCK (1%), and TPCK was 7.18, 9.74, 9.97, and 8.52 days, respectively. The highest mortality (100 % mortality) was observed when a combination of TPCK and SBTI, both at 4% of dietary protein, was used followed by TPCK (4%) that produced 95% mortality. There were significant differences in proteinase activity between treatments and controls when BApNA and SAAPFpNA were used as substrates for trypsin and chymotrypsin, respectively. Reduction of trypsin activity in insects fed with low doses of SBTI (1%), TLCK (1%), and both doses of TPCK (1% and 4%) was 40, 26, 23, and 17%, respectively. Inhibition of chymotrypsin activity was seen in the insects fed on SBTI (1%), TLCK (1%), and TPCK (4%) where inhibition was 14, 9, and 36%, respectively. Maximum inhibition of chymotrypsin activity was observed in the insects fed on diets containing high doses of TPCK (4%). In gel assays, the greatest effects were observed when E. integriceps were fed on high doses of SBTI and TPCK. Therefore, TPCK followed by SBTI proved to be the most effective proteinase inhibitors of E. integriceps.

Trypsin from the viscera of Bogue (Boops boops): isolation and characterisation.

Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS-PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants Km and kcat on BAPNA were 0.13 mM and 1.56 s(-1), respectively, while the catalytic efficiency kcat/Km was 12 s(-1) mM(-1). Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.

TLC-Bioautography as a fast and cheap screening method for the detection of α-chymotrypsin inhibitors in crude plant extracts.

TLC-Bioautography is a fast and effective method for assessing the inhibitory effect of compounds present in plant extracts against microbial species. However, this method has a hidden, currently underutilized potential for evaluating the presence of inhibitory compounds against selected enzymes. The aim of this work was to design a functional TLC-Bioautography method for the evaluation of protease inhibitors present in plant extracts. The method is based on the hydrolysis of Nα-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BApNA) by α-chymotrypsin as a representative serine protease to produce coloured para-nitroaniline (pNA). Derivatization of pNA with both sodium nitrite and N-(1-naphthyl) ethylenediamine (NPED) leads to the formation of a pink azo dye. This step improves the resolution of active compounds on the chromatogram, which appear as light spots on a pink background. The developed method was tested for the analysis of protease inhibitors in different plant materials such as grape pomace from Vitis vinifera, Picea abies bark, Hippophae rhamnoides berries, Hordeum sativum bran, Triticum aestivum bran and Avena sativa bran. Plant extracts, which could not be analysed by a commonly used spectrophotometric method due to interference, were assessed by this method.

Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex.

Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P2(1)), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 A, beta = 96.81 degrees , and diffract X-rays to 1.8 A resolution.

Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin.

PEGylated proteins are routinely used as therapeutics, but systematic studies of the effect of PEG molecular weight and linking chemistry on the biological activity and particularly the thermal stability of the conjugated protein are rarely made. Here, activated monomethoxypolyethylene glycol (mPEG)s (Mw 1100, 2000 and 5000 g/mol) were prepared using succinic anhydride (SA), cyanuric chloride (CC) or tosyl chloride (TC) and used to synthesise a library of trypsin conjugates. The enzyme activity (KM, Vmax and Kcat) of native trypsin and the mPEG-modified trypsin conjugates was compared using N-benzoyl-l-arginine p-nitroanilide (BAPNA) as a substrate, and their thermal stability determined using both BAPNA and N-alpha-benzoyl-l-arginine ethyl ester hydrochloride (BAEE) as substrates to measure amidase and esterase activity respectively. The effect of conjugate chemistry on trypsin autolysis was also examined at 40 degrees C. PEG-trypsin conjugates containing the higher molecular weight of mPEG (5000 g/mol) were more stable than free trypsin, and the conjugate containing CC-mPEG 5000 g/mol had the best thermal stability.

Application and properties of butyl acrylate/pentaerythrite triacrylate copolymers and cellulose-based Granocel as carriers for trypsin immobilization.

The main point was the search for a proper carrier and the kind of carrier activation for trypsin (EC 3.4.21.4) immobilization. The acrylic and cellulose-based carriers were specially prepared in that they possessed the most often used anchor groups: -OH, -NH(2), DEAE and/or -COOH. The immobilization procedures were selected to apply mainly to protein amine groups and appropriate anchor groups on the carrier. As activity tests low (N-benzoyl-dl-arginine-p-nitroanilide, BAPNA) and high (casein) molecular weight substrates were used. It was found, as a rule, that trypsin bound to -COOH groups with the help of carbodiimide was less active and that the amount of bound protein and measured activity (BAPNA) are considerably higher when protein is immobilized via divinyl sulfone. Both rules were observed irrespective of the nature of the polymer matrix. Both types of carriers were found suitable for trypsin immobilization and they were far better than the corresponding Eupergit C-bound enzyme preparations. Taking into account storage stability and activity for both substrates, the divinylsulfone linkage formed between unmodified Granocel and trypsin was the most effective method for the enzyme immobilization. For this preparation, BAPNA and casein conversion, thermal stability at 60 degrees C and estimated kinetic parameters were compared with those obtained for the native enzyme. It was shown that mass transport limitations could be effectively eliminated by suitable conditions and immobilized trypsin was considerably more stable. The values k(cat)/K(m) indicated that the immobilized enzyme was even better as amidase activity was regarded and its potential for protein hydrolysis was only less than twice.

Comparative study of ultrasonic instrumentation for the non-surgical treatment of interproximal and non-interproximal furcation involvements.

BACKGROUND: The aim of this clinical trial was to compare the outcome of non-surgical treatment of interproximal and non-interproximal Class II furcation involvements. METHODS: Thirty-eight patients presenting at least one Class II furcation involvement that bled on probing with a probing depth (PD) > or = 5 mm were recruited. Furcation involvements were grouped as either buccal and lingual furcation involvements (BLFI) or interproximal furcation involvements (IFI). The following clinical outcomes were evaluated: visible plaque index, bleeding on probing (BOP), position of the gingival margin, relative attachment level (RAL), PD, and relative horizontal attachment level (RHAL). N-benzoyl-l-arginine-p-nitroanilide (BAPNA) testing was used to analyze trypsin-like activity in dental biofilm. All parameters were evaluated at baseline and 1, 3, and 6 months after non-surgical subgingival instrumentation. RESULTS: Six months after treatment, both groups had similar means of RAL and RHAL gain (P >0.05). These variables were 1.22 and 1.07 mm in the IFI group and 1.38 and 1.20 mm in the BLFI group, respectively. The PD reduction was significantly greater in the BLFI group than in the IFI group (2.59 and 2.11 mm, respectively; P <0.05). The BLFI group presented fewer sites with PD > or = 5 mm than the IFI group at all post-treatment periods. At 6 months, the BAPNA test showed that only the BLFI group had values significantly different from baseline. This means that the BLFI group had significantly lower BAPNA values compared to the IFI group at 6 months. CONCLUSION: Buccal and lingual Class II furcation involvements respond better to non-surgical therapy compared to interproximal Class II furcation involvements.

Periodontal debridement with povidone-iodine in periodontal treatment: short-term clinical and biochemical observations.

BACKGROUND: The aim of the present study was to evaluate the clinical effects of one-stage periodontal debridement with an ultrasonic instrument, associated with 0.5% povidone (pvp)-iodine irrigation in patients with chronic periodontitis. METHODS: Forty-five patients were randomly assigned into three groups: the control group (CG) received quadrant root planing at 1-week intervals over four consecutive sessions; the periodontal debridement plus pvp-iodine group (PD-PIG) received a 45-minute full-mouth debridement with an ultrasonic instrument, associated with 0.5% pvp-iodine irrigation; and the periodontal debridement group (PDG) received a 45-minute full-mouth periodontal debridement with an ultrasonic instrument, associated with NaCl irrigation. RESULTS: At the 3-month evaluation, the mean probing depth (PD) reduction in CG was 2.51+/-0.52 mm, 2.53+/-0.50 mm in PD-PIG, and 2.58+/-0.60 mm in PDG (P<0.05). The clinical attachment level (CAL) analysis showed a statistically significant gain in all groups compared to baseline (1.87+/-0.56 mm [CG], 1.94+/-0.70 mm [PD-PIG], and 1.99+/-0.92 mm [PDG]). Intergroup analysis of PD and CAL at 1 and 3 months showed no differences (P>0.05). The N-benzoyl-L-arginine-p-nitroanilide (BAPNA) test showed a significant reduction in trypsin activity only during the first month (P<0.05); at 3 months there were no differences compared to baseline (P=0.80). CONCLUSION: This study provides no evidence that pvp-iodine is effective as an adjunct for one-stage periodontal debridement.

Povidone-iodine used as an adjunct to non-surgical treatment of furcation involvements.

BACKGROUND: The aim of this clinical trial was to evaluate the effect of topically applied povidone-iodine (polyvinylpyrrolidone and iodine [PVP-I]) used as an adjunct to non-surgical therapy of furcation involvements. METHODS: Forty-four patients presenting at least one Class II furcation involvement that bled on probing with probing depth (PD)>or=5 mm were recruited. Patients were stratified into two treatment groups: 1) subgingival instrumentation by an ultrasonic device using PVP-I (10%) as the cooling liquid (test); and 2) identical treatment using distilled water as the cooling liquid (control). The following clinical outcomes were evaluated: plaque index, bleeding on probing (BOP), position of the gingival margin, relative attachment level (RAL), PD, and relative horizontal attachment level (RHAL). The N-benzoyl-L-arginine-p-nitroanilide (BAPNA) test was used to analyze the trypsin-like activity in dental biofilm. The clinical and biochemical parameters were evaluated at baseline and 1, 3, and 6 months after therapy. RESULTS: Both groups had similar means of PD reduction and RAL and RHAL gain. At 6 months, these variables were, respectively, 2.31, 1.17, and 1.00 mm in the control group and 2.31, 1.23, and 1.02 mm in the test group. There was also no difference between groups regarding the number of furcation sites presenting RAL gain>or=2 mm. The results of the BAPNA test failed to demonstrate significant differences between groups. CONCLUSION: Non-surgical therapy can effectively treat Class II furcation involvements, and the use of topically applied PVP-I as an adjunct to subgingival instrumentation does not provide additional benefits.

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha).

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors - soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 degrees C and was stable from pH 4.0 to pH 10.0 when incubated at 20 degrees C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of DL-BAPNA by the chinook salmon enzyme was 60 degrees C, however, the enzyme was unstable at temperatures above 40 degrees C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS-PAGE.

Trypsin and antitrypsin activities and protein concentration in serial meconium and feces of healthy newborns.

BACKGROUND: Meconium has the potential for being the matrix on which markers of fetal exposure to physiologic and non-physiologic agents during intrauterine life can be analyzed. The aim of this study was to compare trypsin and antitrypsin activities and protein concentration during intra- and extrauterine human development based on an assessment of these parameters in serial meconium and first feces of healthy, term newborns during the first four days of life. METHODS: One hundred and eighteen mature, term newborns were studied. Single portions of meconium or feces were taken prospectively from day 1 to day 4. In ten newborns each meconium and feces passed by the infant from birth up to the fourth day after delivery was collected individually. Trypsin activity was measured using L-TAPA (N-alpha-tosyl-l-arginine-p-nitroanilide) as substrate, trypsin inhibitory capacity (TIC) with N-alpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate, and protein concentration by the method of Lowry et al. RESULTS: Meconium and feces of healthy newborns demonstrated a decrease in TIC during the first two days of life and an increase in trypsin activity over the first three successive days of life. No correlation was observed between the variability of both parameters. During the first three days of a newborn's life, significant correlation was observed between TIC and protein concentration in meconium and first feces. In the course of proteolytic activity changes in successive portions of meconium and feces, a transient peak occurs on the 2nd-4th day of life. CONCLUSIONS: The gradual decrease in protease inhibitor activity and low trypsin activity in successive portions of meconium from the first two days of extrauterine life may provide a retrograde chronological depiction of the course of the second and third trimesters of intrauterine life. Serial collection of feces over 2-4 days may provide a reflection of the dynamics of the postnatal increase in the newborn's pancreatic exocrine functions.

Trypsin inactivation by latex fabricated gold nanoparticles: A new strategy towards insect control.

Before applying nanotechnologies in biomedical and environmental areas it is advised to study interactions of nanoparticles and other nanomaterials with biomacromolecule present in living system. Moreover there is scarcity of reports on interactions between nanoparticles and biomaterials. In present report a rapid, ecofriendly method of fabricating stable gold nanoparticles (AuNPs) using latex of Jatropha curcas is reported for the first time. AuNPs found to have characteristic absorption maxima centered at 540nm, multiple irregular shapes with size range from 20 to 50nm and have crystalline nature. Latex fabricated AuNPs were found to inhibit catalytic potential of trypsin (a vital enzyme responsible for digestion, insecticide resistance and in several disease conditions). The interactions between AuNPs and trypsin were analyzed by UV-vis spectrophotometry and microwave plasma-atomic emission spectrometry which suggests formation of trypsin-AuNPs complex responsible for lowering catalytic activity of trypsin. Transmission electron microscopy, Fourier transform infrared spectroscopy and particle size distribution studies further confirm complex formation between trypsin and AuNPs. Diverse interactions of metal nanoparticles with proteins such as covalent interaction, electrostatic interactions and binding to SH group of amino acid may be the reasons behind inhibition of trypsin activity. In vivo studies on serum of several vectors and agriculturally important pests supported instrumental results on AuNPs induced trypsin inhibition. This work will bring a new research direction to explore eco-friendly nanoparticle in insect control via inhibition of enzyme catalytic potential.

Evaluation of the toxicity of ionic liquids on trypsin: A mechanism study.

The toxicity of ionic liquids (ILs) was evaluated by using trypsin as biomarker. Experimental results indicated that the trypsin activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. Primary analysis illustrated that hydrophobicity of ILs was one of the driven forces ruling the ILs-trypsin interaction. Thermodynamic parameters, Gibbs free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of trypsin in the presence of ILs. Both negative ΔH and ΔS suggested hydrogen bonding was the major driven force underlying the IL-trypsin interaction. To assess the toxicity of ILs, it should be considered the combination of the hydrogen bonding ability and hydrophobicity of ILs. A regression based model was established to correlate the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs.