Bovine Trypsin Research Articles

Proteinase inhibitors of pigeonpea cv. BSMR 736: Characterization and bioefficacy against Helicoverpa armigera

Pigeonpea is an agriculturally important leguminous crop with high vulnerability to insect pest attack specifically, Helicoverpa armigera. The proteinase inhibitors (PIs) mediated host plant resistance against insect pests is a promising sustainable agricultural research practice. The current study was carried out to perceive biochemical characterization of proteinase inhibitors named PPTI in the pigeonpea (cv. BSMR 736). The purification of PPTI from crude protein seed extract was achieved by acetone precipitation, N-LP-IEF, and trypsin affinity chromatography. It was found to inhibit bovine trypsin and HaGPs in vitro. The optimal conditions for inhibition were pH 8 and temperature 40ºC. The PPTI showed four isoinhibitors bands on native, non-reducing and reducing SDS-PAGE in the range of 26.7–19.3 KDa. Upon resolution on two-dimensional gel electrophoresis (2-DE), PPTI produced nine pI variant spots having isoelectric point (pI) 6.6, 6.6, 6.3, 6.1, 5.9, 5.8, 5.7, 5.6 and 5.6. An artificial diet containing PPTI reduced the H. armigera larval weight about 69%, with 25% mortality. For eco-friendly sustainable agricultural practices, natural compounds like PPTI could be expressed in transgenic crops to prevent the invasion of H. armigera in pigeonpea.

Evaluation of prime and prime-boost immunization strategies in BALB/c mice inoculated with Leishmania infantum transfected with toxic plasmids

The etiologic agents of visceral leishmaniasis are Leishmania infantum and Leishmania donovani. Despite the variety of drugs available to treat leishmaniasis, most lead to serious adverse effects, and resistance to these drugs has been reported. Currently, no leishmaniasis vaccine is available for humans. That is why the group developed transgenic L. infantum promastigote lines, which express toxic proteins after differentiation into amastigotes. That is why group developed the pFL-AMA plasmid and transfected it into L. Infantum promastigotes. This plasmid was expressed only in the amastigote form of the parasite. Sequences encoding toxic proteins (active bovine trypsin and egg avidin) were inserted in this plasmid, and the transfected parasites died after the differentiation process. In this study, two immunization protocols were performed in BALB/c mice: prime and prime-boost immunization prior to challenge with the wild-type L. infantum (WT). The parasite burdens in the spleen, liver, and bone marrow were evaluated to verify immunological protection. Histopathological analysis of the spleen and liver and the humoral immune response were also performed. The data showed that the parasite burden was reduced in prime-boosted mice in the spleen, liver, and bone marrow, indicating that mice immunized with two doses of the transfected parasites were satisfactorily protected. High levels of IgG, IgG1, and IgG2a antibodies were observed, as well as the presence of anti-inflammatory cytokine Interleukine-10 and pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN – γ) suggesting a Th1/Th2 mix response, in addition to the presence of multinucleated giant cells in the spleen and lymphocyte infiltration in the liver. Therefore, L. infantum transfected with a toxic plasmid is an excellent vaccine candidate against visceral leishmaniasis and the application of a boost before the challenge promotes greater protection against WT L. infantum infection.

Silver nanoprism-mediated protein estimation—an ultrasensitive platform for rapid estimation of protein concentration

Estimation of protein concentration in the range of nanogram level (ng/ml) is a big challenge for conventional protein estimation methods. The highly dipole sensitive spectroscopic properties of Silver nanoprism (AgNPR) has been utilized to develop a rapid and highly sensitive method for the estimation of globular protein concentration at ng/ml (or ppb) range. We have applied a unique molecular doping approach to introduce protein in the interstitial space of the Ag fcc(111) crystal planes within AgNPR structure. The presence of the doped protein induces deformation in the crystal plane arrangement of AgNPR that results in a quantitative red shift of the dipole resonance peak (D-peak) of AgNPR under UV–vis spectroscopy. The proposed method allows detection of a protein concentration range of as low as 1–20 ng ml−1- that is better than the sensitivity limit of conventional protein estimation techniques. This method has been successfully applied for commonly used proteins like haemoglobin (Hb), Bovine serum albumin (BSA), Trypsin (TRYP) and Lysozyme (LYS) with a very low limit of detection (LOD) within 2–6 ng ml−1. The lowest LOD value was shown by Hb as 2.08 ng ml−1. The method has further been validated by measuring Casein concentration from milk with an accuracy of 99% and 95% recovery for the concentration of 3.1 and 31 ng ml−1 respectively. Transmission emission microscopy (TEM) images show that the doped protein has been found to alter the size and shape of the AgNPR as a function of the dopant concentration by creating systematic deformation. This method does not require any alteration of the reaction temperature and solely depends on the physical interaction of doped protein with its neighbouring crystal structure of the nanoplanar geometry.

Natural v/s Commercial trypsin isolated from liver waste of Labeo rohita

The mounting human population and the massive amount of waste generated from the same is receiving particular attention towards valorisation of waste. According to the annual report of FAO, the human consumption of fish protein has reached 87% in 2016 from 67% during 1960s [1]. Aquaculture only has contributed to 5.8% annual growth rate among food sectors in the past decade. In this milieu, disposal of fish visceral waste is becoming a major menace to fishery industries exerting a great economic and environmental impact. Being perishable in nature, the organic portion of the waste decomposes rapidly and acts as a breeding ground for microbes. Moreover, the hefty and indiscriminate use of antibiotics and disinfectants in farmed animals is developing resistant strains, thus raising environment and ecological concerns. In order to solve such problem, the present investigation focussed upon employing the visceral trypsin as a cell dissociating agent. The efficacy of trypsin obtained from viscera of Labeo rohita upon KB cell line (Doubling time 50 hrs.) was assessed in terms of cell viability. The cytotoxic effect of the visceral trypsin at 0.01%, 0.1% and 1% concentration were investigated at three time points (10 sec, 15 sec and 20 sec). Commercial (bovine) trypsin was considered as control. A time dependent decrease in cell viability upon gradually increasing the concentration was observed in all groups of treatment. The lowest reduction in cell viability (2%) was observed with 1% concentration at 15 sec and 20sec. Although, commercial trypsin was found more efficient than trypsin isolated from waste during this study but the potency of visceral trypsin observed cannot be ruled out. Thus, the application of this enzyme as a cell-dissociating agent suggested it as a comparable candidate with commercial trypsin.

Novel proteinase inhibitor from the hemolymph of soybean pest Anticarsia gemmatalis (lepidóptera: Noctuidae): Structural modeling and enzymatic kinetic.

New approaches are needed to reduce risks to the environment and natural enemies and to avoid or delay the onset of insecticide resistance. The use of insecticides based on proteinase inhibitors of hemolymph is an alternative for the control of Lepidoptera pests primarily by having low toxicity and short persistence in the environment. Thus, in this study, we describe the purification process and identification of protease inhibitors from hemolymph Anticarsia gemmatalis and their activities against trypsin enzymes. Furthermore, the three-dimensional (3D) structure of the inhibitor and binding mode to trypsin enzymes was determined, and the stability of the inhibitory activity in several pHs and temperature values was evaluated. The inhibitor was characterized as a serpin family inhibitor and named A. gemmatalis hemolymph serpin inhibitor (AHSI), with an approximate mass of 38 ± 2 kDa, highly stable to temperature and pH variations, and with inhibitory capacity on bovine trypsin and gut trypsin of A. gemmatalis demonstrated by calculated Ki values and affinity energy through molecular docking, being a reversible competitive inhibitor that binds to the active site of trypsin-like enzymes. We conclude that the AHSI inhibitor identified from the hemolymph of the soybean pest A. gemmatalis preserves the original structure of the serpin family with a good overall stereochemical quality confirmed from molecular modeling. The docking analysis showed that the reactive site of the inhibitor is in contact with the catalytic cavity of the trypsin with high-affinity energy.

Metagenomic analysis reveals presence of different animal viruses in commercial fetal bovine serum and trypsin.

Animal-derived biological products, such as fetal bovine serum (FBS) and trypsin, are important supplements for scientific, pharmaceutical, and medical use. Although preventive guidelines and tests are implemented to reduce potential viral contamination in these biologicals, they do not target unusual or emerging viruses, leading to safety concerns. Using unbiased metagenomics, we investigated the presence of viruses in recently collected commercial FBS and trypsin samples from different geographic regions. In total, we detected viral sequences belonging to Parvoviridae,Anelloviridae,Flaviviridae, Herpesviridae,Caliciviridae,Nodaviridae,Rhabdoviridae, and Paramyxoviridae, including several viruses related to bovine diseases, viruses of potential human and insect origin, and viruses of unknown origin. Bovine parvovirus 3 and bosavirus were detected with high frequency and abundance in FBS, necessitating more stringent testing for these parvoviruses during production. Both bovine norovirus and bovine viral diarrhea virus 1 displayed relatively high genetic distance to closest hits, indicating the presence of new genotypes in farm animals. While the origin of novel lyssavirus and Nipah virus is unclear, their presence raises the possibility of the introduction of pathogenic animal-derived viruses into biologicals. Our results showed relatively widespread contamination of different viruses in biologicals, underscoring the need for robust safety protocol alternatives, such as metagenomic sequencing, to monitor emerging viruses.

Atividade inseticida de um inibidor de peptidase das sementes de Anadenanthera macrocarpa contra Anagasta kuehniella

ABSTRACT Protease inhibitors (PIs) are a part of the plant defense system and reduce the proteolytic activity of the digestive enzymes of insect pests. The current study aimed to isolate and characterize an inhibitor of trypsin (AmTI) within the seed of Anadenanthera macrocarpa (Benth) (Leguminosae-Mimosoideae). Moreover, we tried to assess the defense mechanism of the larvae of Anagasta kuehniella against this inhibitor. Protein seed extracts were purified using Sephadex G-50 and trypsin-Sepharose columns. Electrophoresis revealed the molecular weight of the inhibitor to be 25 kDa. The stability evaluation demonstrated that the inhibitor was not denatured at temperatures of up to 60 °C, pH 2-10, and concentrations of up to 100 mM dithiothreitol for one hour. The inhibitor reacted in a 1:1 ratio with bovine trypsin with an inhibition constant [Ki] = 2.517 ´ 10-8. Incorporating the inhibitor in a 1 mg per 100 mg proportion of artificial diet offered to A. kuehniella larvae led to a significant difference in the weight and survival of larvae of the fourth instar compared to the control. AmTI acted on the enzymatic activities of trypsin and chymotrypsin, not allowing until the fourth larval instar, A. kuehniella, to create adaptations against the inhibitor, as it had a simultaneous effect on larval weight and mortality. Therefore, a new trypsin inhibitor showing inhibitory activity against the digestive enzymes trypsin and chymotrypsin from A. kuehniella was isolated, indicating that these activities are correlated with the deleterious effects of this insect.

Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI.

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191-Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4-BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

Relative Mobility (Rf) Analysis of Albumin Isolates from Snakehead Fish (Ophiocephalus striatus) Extracted at Different Temperatures and Times

Snakehead fish was a fish that has a high albumin content. Snakehead fish was used in the health sector known as medicinal freshwater fish, to accelerate the process of healing wounds after surgery and childbirth. The purpose of this study was to analyze the relative mobility (Rf) of the snakehead fish (Ophiocephalus striatus) albumin isolates by steaming at different temperatures and times. The research method for analyze the relative mobility (Rf) of the snakehead fish (Ophiocephalus striatus) albumin isolates by using gel filtration column chromatography, was detected by the SDS-PAGE electrophoresis method. Electrophoresis was carried out at a constant voltage of 125 v/ slab. Gel staining was carried out with Commasie Brilliant Blue R-250 in methanol-acetic acid-water. A standard protein mixture consisting of: myosin (200.0 kDa), β-galactosidase (116.3 kDa), phosphorylase B (97.4 kDa), bovine serum albumin (66, 2 kDa), ovalbumin (45.0 kDa), carbonic anhydrose (31.0 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and aprotinim (6.5 kDa). The data analysis of this research was descriptive analysis to see the photos of the electrophoresis results. Meanwhile, for the analysis, the measurement of albumin isolation by gel filtration Sephadex G-75 was carried out with a divided plot design. The results showed that SDS-PAGE electrophoresis with the most complex amount of protein was albumin isolate, the effect of the steaming temperature was 40 oC for 30 minutes, located in 5 ml of the 1st fraction, 5 ml of the 2nd fraction and 5 ml of the 3rd fraction, with values of relative mobility (Rf) 0.0833 to 0.6944.

Estimating the probability of coincidental similarity between atomic displacement parameters with machine learning

High-resolution diffraction studies of macromolecules incorporate the tensor form of the anisotropic displacement parameter (ADP) of atoms from their mean position. The comparison of these parameters requires a statistical framework that can handle the experimental and modeling errors linked to structure determination. Here, a Bayesian machine learning model is introduced that approximates ADPs with the random Wishart distribution. This model allows for the comparison of random samples from a distribution that is trained on experimental structures. The comparison revealed that the experimental similarity between atoms is larger than predicted by the random model for a substantial fraction of the comparisons. Different metrics between ADPs were evaluated and categorized based on how useful they are at detecting non-accidental similarity and whether they can be replaced by other metrics. The most complementary comparisons were provided by Euclidean, Riemann and Wasserstein metrics. The analysis of ADP similarity and the positional distance of atoms in bovine trypsin revealed a set of atoms with striking ADP similarity over a long physical distance, and generally the physical distance between atoms and their ADP similarity do not correlate strongly. A substantial fraction of long- and short-range ADP similarities does not form by coincidence and are reproducibly observed in different crystal structures of the same protein.

Adaptation of the HEp-2 cell line to totally animal-free culture systems and real-time analysis of cell growth.

Routine cell culture demands the use of animal-derived products, mainly fetal bovine serumand swineor bovine trypsin. According to the 3Rs principle and to theEuropean Centre for the Validation of Alternative Methods, animal-free substitutes are strongly recommended for in vitro methods. In this study, theHEp-2 cell line was adapted to different totally animal-free culture systems, such as a serum-free complete medium (VP-SFM), human platelet lysate and a synthetic trypsin (TrypLE™ Express); afterward, cell growth was assessed with the xCELLigence instrument. Animal-free products provided promising results, with performances similar or preferable to the common reagents; thereforetheir use could be encouraged for bothethical and technical advantages.

An International Collaborative Study on Trypsin Inhibitor Assay for Legumes, Cereals, and Related Products

AbstractFor determining trypsin inhibitor activity (TIA) in soy products, the American Oil Chemists' Society (AOCS) Method Ba 12‐75 has been used. It measures differences in absorbance at 410 nm of bovine trypsin activity toward a synthetic substrate (Nα‐benzoyl‐DL‐arginine‐p‐nitroanilide) in the absence and presence of an inhibitor. Recently, a significantly improved method was developed (JAOCS, 2019, 96:635–645), featuring 5 mL of total assay volume, enzyme‐last sequence, and single inhibitor level in duplicate. It is proposed as the AOCS Method Ba 12a‐2020. As a part of the AOCS method approval process, a collaborative study involving 12 international laboratories was conducted to evaluate the performance of the proposed method. The study involved measuring TIA in 10 selected test samples plus a blind duplicate. They included soybeans, pulses, cereals, and their processed products (flours, concentrates, and isolates). After rigorous statistical treatment of the data, only three outliers were removed from the data of two samples. Repeatability relative standard deviations (RSDr) for the 11 samples ranged from 0.99% to 5.52%. Reproducibility RSD (RSDR) ranged from 7.07% to 22.92%, with seven samples having RSDR around 10% or less. The remaining four samples had very low TIA, and their RSDR values ranged from 13.34% to 22.92%. The study has demonstrated reliable performance of the proposed AOCS method. Several collaborators carried out additional experiments addressing some aspects of the method, leading to further refinements. The proposed method is undergoing evaluation by the AOCS Uniform Methods Committee for adoption as an Official Method for measuring TIA in various legume and grain products.

Molecular characterization of diverse defensins (γ-thionins) from Capsicum annuum flowers and their effects on the insect pest Helicoverpa armigera

Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) constituting the host defense system. They are known to interact with the cell membranes to exhibit antifungal and antibacterial activity. Though defensin genes are inducible under biotic and abiotic stress, their involvement in insect herbivore defense is not well studied. In this work we studied the phylogeny, structural diversity, biochemical and functional properties of defensins from Capsicum annuum L with particular emphasis on their interaction with insect and fungal pathogens. Phylogenetic analysis of defensin peptides from Solanaceae, Pinaceae, Fabaceae, Asteraceae, Brassicaceae and similar peptides from invertebrates divided them into ten groups and Capsicum peptides were represented in most of them. Three thionin (CanThio-1, −2, −3) and two defensin (CanDef-20, −21) genes isolated from the flowers of Capsicum annuum were cloned and recombinant proteins were expressed. The mature peptide amino acid sequences of CanThio and CanDef were 54–75 aa with 21.82 to 73.81% similarity. They contained the conserved features of defensin peptides like the gamma core region and the presence of eight cysteine residues. Predicted structural alignment indicated that CanThio and CanDef had structural diversity and structurally unaligned loop regions.The recombinant CanThio and CanDef inhibited 5% - 65% Aspergillus oryzae amylase activity at 20 μM and ~ 20% of Helicoverpa armigera gut amylase activity at 50 μM peptide concentrations. CanThio-2 displayed bovine trypsin inhibition (~14%) while all the CanThio and CanDef inhibited ~20% of H. armigera gut proteinase activity at 20 μM. H. armigera larvae fed on an artificial diet incorporated with recombinant CanThio-2 and CanDef-20 (125 μg/ml) exhibited a 15% reduction in larval mass and 13% reduction in pupal mass. The CanThio and CanDef peptides also exhibited antifungal properties against pathogenic fungus Fusarium oxysporum at 5 μg/ml, where CanThio-2 and CanDef-20 showed the strongest activity, which may be attributed to the positively charged amino acids in the γ-core region.We conclude that despite having relatively similar conserved three-dimensional structures CanThio and CanDef display functional variability, which can be utilized for enhancing insect and fungal resistance in plants.

Controlling the kinetics of visible-light-induced photocatalytic performance of gold decorated graphitic carbon nitride nanocomposite using different proteins

In this work, the interaction of different proteins with two dimensional (2D) graphitic carbon nitride (GCN) nanosheets decorated with gold nanoparticles (Au NP) is reported along with their influence in controlling the visible-light-induced photocatalytic activity. The decoration of Au NP on 2D GCN nanosheets improves the photocatalytic activity as Au NP plays an important role in trapping and storing the conduction band electrons of GCN nanosheets. The structural, morphological and optical properties of Au-GCN (AGCN) nanocomposite have been investigated using scattering and spectroscopic techniques. Methylene blue (MB) dye was chosen as a representative system for the studying of visible-light-induced photocatalytic activity of AGCN nanocomposite in the presence of different proteins. Bovine serum albumin (BSA), trypsin and cytochrome C having different chain lengths and physicochemical properties were used to control the kinetics of the photocatalytic activity of GCN and AGCN nanocomposites. Due to the simultaneous competitive adsorption of MB and proteins, the photocatalytic activity of the nanocomposites got enhanced in the presence of BSA, while it got inhibited or slowed down in the presence of trypsin and cytochrome C, respectively. The observed photocatalytic activity could be correlated with the surface charges present on the proteins and the mechanistic understanding developed in this study can be beneficial in controlling the kinetics of the photocatalytic activity of semiconductor-based photocatalysts using proteins as activators or inhibitors.

High-resolution macromolecular crystallography at the FemtoMAX beamline with time-over-threshold photon detection.

Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, Rmerge/Rmeas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.

Structural modelling and thermostability of a serine protease inhibitor belonging to the Kunitz-BPTI family from the Rhipicephalus microplus tick

rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.

Different electrically charged proteins result in diverse transport behaviors of plastic particles with different surface charge in quartz sand

The influence of proteins on the transport and deposition behaviors of microplastics (MPs) in quartz sand was examined at both low (5 mM) and high ionic strength (25 mM) in NaCl solutions at pH 6. Carboxylate- and amine-modified polystyrene latex microspheres with size of 200 nm were employed as negatively (CMPs) and positively surface charged MPs (AMPs), respectively, while bovine serum albumin (BSA) and bovine trypsin were utilized as representative negatively and positively charged proteins, respectively. The results showed that for two examined protein concentrations (both 1 and 10 mg/L TOC) under both ionic strength conditions, the presence of BSA increased the transport of both CMPs and AMPs, while the presence of trypsin decreased the transport of CMPs yet increased the transport of AMPs in porous media. The mechanisms driving to the changed transport of MPs induced by two types of proteins were found to be different. Particularly, steric interaction induced by BSA corona adsorbed onto CMPs surface as well as the repel effects resulted from BSA suspending in solutions were found to contribute to the enhanced CMPs transport with BSA copresent in suspensions. The increased sizes and the decreased electrostatic repulsion of CMPs due to the adsorption of trypsin onto CMPs, together with the addition of extra deposition sites due to the adsorption of trypsin onto quartz sand drove to the decreased CMPs transport with trypsin copresent in suspensions. The increased electrostatic repulsion due to the adsorption of BSA onto AMPs surfaces caused the enhanced AMPs transport with BSA in solutions. While, the decreased electrostatic attraction of AMPs due to the adsorption of trypsin onto AMPs, as well as the competition of deposition sites due to the adsorption of trypsin onto quartz sand contributed to the increased AMPs transport with trypsin copresent in suspensions. The results showed that the presence of different types of proteins would induce different transport behaviors of microplastics with different surface charge in porous media. Since proteins are widely present in aquatic systems, to more accurately predict the fate and transport of MPs in natural environments, the effects and mechanisms of proteins on the transport of MPs should be considered.

Trypsin inhibitor activity in representatives of the genus Hedysarum (Fabaceae): the polyvariance of seasonal dynamics in Southern Siberia

Background. Trypsin-inhibiting activity (TIA) is one of the factors of plant immunity. This factor provides the presence of general non-specific systemic resistance. Representatives of the genus Hedysarum L. are promising medicinal and high-protein fodder plants with high TIA in the leaves. High TIA values in the aboveground part of plants lead to a decrease in protein assimilation by animals. Therefore, identifying the differentiation in the features of TIA seasonal dynamics will make it possible to use the potential of these species most effectively under the conditions of Southern Siberia.Materials and methods. TIA in the leaves of mature generative plants of Hedysarum theinum Krasnob. and H. flavescens Regel & Schmalh. was analyzed. H. theinum is a rare species for Siberia; H. flavescens is a settled one in the forest-steppe zone of Western Siberia. The studies were conducted with H. theinum in 2010 under natural conditions (Altai Republic) and in cultivation (forest zone of Western Siberia). In 2017, H. theinum and H. flavescens were studied under cultivation in the forest-steppe zone of Western Siberia. The research method was based on the spectrophotometric measurement of the optical density in protein substrate (BAPA) decomposition products under the effect of trypsin (at 405 nm). Bovine trypsin manufactured by ISNBiomedical (USA) was used. BAPA (Na -benzoyl-DL-argininep-nitroanilide) served as a substrate.Conclusions. The TIA values in the flowering phase of seasonal plant development reached their maximum only under favorable conditions: in Altai Republic for the leaves of H. theinum, and in the foreststeppe zone of Western Siberia for H. flavescens. TIA values in H. theinum did not increase during flowering in the forest zone of Western Siberia and significantly decreased in the forest steppe. In view of these findings, these two species may be recommended for practical utilization in this phase of seasonal development.

PARAFAC study of L-cys@CdTe QDs interaction to BSA, cytochrome c and trypsin: An approach through electrostatic and covalent bonds

Utilizing fluorescence spectroscopy, non-covalent and covalent interactions of L-cys@CdTe quantum dots to bovine serum albumin (BSA), cytochrome c and trypsin were investigated. L-cys@CdTe QDs with the emission maximum at 530nm and an average diameter of 2.6nm were synthesized in the aqueous medium. Formaldehyde, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) with N-hydroxysuccinimide (NHS), and glutaraldehyde was applied as cross-linkers. In the case of both electrostatic and covalent strategies PARAFAC, as a powerful multi-way chemometrics technique, was utilized to analyze fluorescence excitation-emission (EEM) spectra. For non-covalent and covalent bonding, two and three significant components composed the PARAFAC models. Resolved EEM shows that in the presence of formaldehyde, a new component with an emission peak similar to BSA was obtained. Using EDC-NHS cross-linker, the fluorescence peak of the newly formed component was in a distinct wavelength with similar emission intensity, compared to L-cys@CdTe QDs and BSA. Employing glutaraldehyde, a distinguished component was easily detected at emission wavelengths higher than that of L-cys@CdTe QDs and proteins. It was concluded that the choice of cross-linker is a critical step to create different emission spectra when dealing with nano-bio-conjugations. This study shows that glutaraldehyde cross-linker leads to increase sensitivity, selectivity, and accuracy of protein analysis.

The Energetic Origin of Different Catalytic Activities in Temperature-Adapted Trypsins.

Psychrophilic enzymes were always observed to have higher catalytic activity (kcat) than their mesophilic homologs at room temperature, while the origin of this phenomenon remains obscure. Here, we used two different temperature-adapted trypsins, the psychrophilic Atlantic cod trypsin (ACT) and the mesophilic bovine trypsin (BT), as a model system to explore the energetic origin of their different catalytic activities using computational methods. The results reproduce the characteristic changing trends in the activation free energy, activation enthalpy, and activation entropy between the psychrophilic and mesophilic enzymes, where, in particular, the slightly decreased activation free energy of ACT is determined by its considerably reduced activation enthalpy rather than by its more negative activation entropy compared to BT. The calculated electrostatic contributions to the solvation free energies in the reactant state/ground sate (RS/GS) and transition state (TS) show that, going from BT to ACT, the TS stabilization has a predominant effect over the RS stabilization on lowering the activation enthalpy of ACT. Comparison between the solvation energy components reveals a more optimized electrostatic preorganization to the TS in ACT, which provides a larger stabilization to the TS through reducing the reorganization energy, thus resulting in the lower activation enthalpy and hence lower activation free energy of ACT. Thus, it can be concluded that it is the difference in the protein electrostatic environment, and hence its different stabilizing effects on the TS, that brings about the different catalytic activities of different temperature-adapted trypsins.