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Journal of Biochemistry Nov 1982We devised a new separation technique for the protein, "affinophoresis," which is based on its specific affinity and utilizes electrophoresis. This technique requires a...
We devised a new separation technique for the protein, "affinophoresis," which is based on its specific affinity and utilizes electrophoresis. This technique requires a carrier macromolecule, "affinophore," which contains both an affinity ligand for a certain protein and many charges, either positive or negative, in order to migrate rapidly in an electric field. When a mixture of proteins is electrophoresed in the presence of the affinophore, the protein having an affinity with the ligand will form a complex with the affinophore. This results in a change in the apparent electrophoretic mobility. If the protein is sufficiently accelerated, we can separate it from other materials. A cationic affinophore for trypsin was prepared. Soluble dextran MW approximately 10,000) was coupled with a DEAE-group and m-aminobenzamidine, a competitive inhibitor of trypsins. Electrophoresis of trypsins from several origins on agarose gel plates in the presence of the affinophore showed that affinophoresis actually occurred. The electrophoretic mobilities of trypsins increased towards the cathode, the same direction as the affinophore movement. The presence of leupeptin and treatment of the trypsins with TLCK suppressed the effect of the affinophore. Streptomyces griseus trypsin, contained in Pronase, was easily separated and detected. This procedure is distinct from affinity chromatography and so-called affinity electrophoresis in that the support of the affinity ligand moves, and has advantages especially for analytical purposes: for example, the detection of specific molecules regardless of their isoelectric points.
Topics: Affinity Labels; Aminocaproates; Animals; Cattle; Chromatography, Affinity; Dextrans; Electrophoresis; Ethanolamines; Indicators and Reagents; Trypsin
PubMed: 6185472
DOI: 10.1093/oxfordjournals.jbchem.a134087 -
American Journal of Physiology.... Nov 2004Sphingomyelin (SM) hydrolysis in the gut has implications in colonic tumorigenesis and cholesterol absorption. It is triggered by intestinal alkaline sphingomyelinase...
Sphingomyelin (SM) hydrolysis in the gut has implications in colonic tumorigenesis and cholesterol absorption. It is triggered by intestinal alkaline sphingomyelinase (Alk-SMase) that is present in the intestinal mucosa and content. The mechanism by which the enzyme is released into the lumen is not clear. We studied whether trypsin can dissociate Alk-SMase from the mucosa and affect its activity. During luminal perfusion of rat intestine, addition of trypsin to the buffer increased Alk-SMase activity in the perfusate output by about threefold. Treating COS-7 cells transfected with Alk-SMase cDNA with trypsin increased the SMase activity in the medium and reduced that in the cell lysate dose dependently. The appearance of Alk-SMase in the perfusate and culture medium was confirmed by Western blot analysis. The effect of trypsin was blocked by trypsin inhibitor, and neither chymotrypsin nor elastase had a similar effect. We also expressed the full length and COOH-terminal truncated Alk-SMase in COS-7 cells and found that the activity of the full-length enzyme is mainly in the cells, whereas that of the truncated form is mainly in the medium. Both forms were active, but only the activity of the full-length Alk-SMase was enhanced by trypsin. By linking a poly-His tag to the constructed cDNA, we found that the first tryptic site Arg440 upstream of the signal anchor was attacked by trypsin. In conclusion, trypsin cleaves the Alk-SMase at the COOH terminal, releases it from mucosa, and meanwhile enhances its activity. The findings indicate a physiological role of trypsin in SM digestion.
Topics: Alkalies; Animals; COS Cells; Chlorocebus aethiops; Dose-Response Relationship, Drug; Intestinal Mucosa; Intestines; Pancreas; Peptide Fragments; Rats; Rats, Sprague-Dawley; Sphingomyelin Phosphodiesterase; Transfection; Trypsin
PubMed: 15205117
DOI: 10.1152/ajpgi.00190.2004 -
Nature Communications Sep 2022The process of ligand-protein unbinding is crucial in biophysics. Water is an essential part of any biological system and yet, many aspects of its role remain elusive....
The process of ligand-protein unbinding is crucial in biophysics. Water is an essential part of any biological system and yet, many aspects of its role remain elusive. Here, we simulate with state-of-the-art enhanced sampling techniques the binding of Benzamidine to Trypsin which is a much studied and paradigmatic ligand-protein system. We use machine learning methods to determine efficient collective coordinates for the complex non-local network of water. These coordinates are used to perform On-the-fly Probability Enhanced Sampling simulations, which we adapt to calculate also the ligand residence time. Our results, both static and dynamic, are in good agreement with experiments. We find that the presence of a water molecule located at the bottom of the binding pocket allows via a network of hydrogen bonds the ligand to be released into the solution. On a finer scale, even when unbinding is allowed, another water molecule further modulates the exit time.
Topics: Benzamidines; Ligands; Protein Binding; Proteins; Trypsin; Water
PubMed: 36114175
DOI: 10.1038/s41467-022-33104-3 -
European Journal of Biochemistry Apr 1996Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reversed transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and...
Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reversed transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid-amplification-of-cDNA-ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophilia trypsin were built and compared. Both models contain two domains of beta-barrel sheets as was shown by means of X-ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp176 sits as the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugar-fed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10(-9) M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Complementary; Diptera; Female; Humans; Insect Hormones; Male; Models, Molecular; Molecular Sequence Data; Oligopeptides; Protein Biosynthesis; RNA, Messenger; Sequence Homology, Amino Acid; Trypsin
PubMed: 8620885
DOI: 10.1111/j.1432-1033.1996.0279n.x -
Journal of Proteome Research Apr 2009Among trypsin family proteases, bovine and porcine trypsins are currently the enzymes of choice for proteomics applications. However, there are trypsins from other...
Among trypsin family proteases, bovine and porcine trypsins are currently the enzymes of choice for proteomics applications. However, there are trypsins from other sources that have higher catalytic activities than mammalian trypsins. Of these, Streptomyces erythraeus trypsin (SET) is particularly attractive, because SET has more than 1 order of magnitude greater amidase activity than mammalian trypsin and is resistant to autolytic degradation. These properties are advantageous for many proteomics applications. To evaluate this protease for proteomic applications, we expressed SET in E. coli, purified it to homogeneity, and then examined its enzymatic properties. As expected, recombinant SET (rSET) had greater than an order of magnitude higher amide bond hydrolysis activity (Km/k(cat)) for both N(alpha)-benzoyl-L-arginine-p-nitroanilide and N(alpha)-benzoyl-L-lysine-p-nitroanilide than modified porcine trypsin and did not show any sign of autolytic degradation after 96 h of incubation at 37 degrees C. The performance of rSET for proteomic applications was evaluated by applying the protease for in-solution and in-gel digestion of bovine serum albumin, and for 18O labeling of peptides. These results confirmed that rSET has the potential to be a useful protease in such proteomic experiments. We also report various properties of rSET that are fundamental to the use of this protease for proteomics applications.
Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Molecular Sequence Data; Proteomics; Saccharopolyspora; Substrate Specificity; Swine; Trypsin
PubMed: 19231893
DOI: 10.1021/pr8004919 -
Sheng Li Xue Bao : [Acta Physiologica... Dec 2022In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test,...
In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.
Topics: Humans; Trypsin; Alkaloids
PubMed: 36594389
DOI: No ID Found -
PloS One 2020Tryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and...
Tryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and biopharmaceutical product characterization, owing to the high level of cleavage fidelity produced with this technique. However, nonspecific trypsin cleavages have been frequently reported and shown to be related to a number of digestion conditions and predigestion sample treatments. In this work, we reveal that, for a number of commercial trypsins, reconstitution and storage conditions can have a significant impact on the occurrence of trypsin nonspecific cleavages. We analyzed the tryptic digestion of a variety of biotherapeutics, using trypsins reconstituted under different conditions. The results indicate that, for many commercial trypsins, commonly recommended reconstitution/storage conditions (mildly acidic, e.g., 50 mM acetic acid, 1 mM HCl) can actually promote nonspecific trypsin activities, which are time dependent and can be as high as 20% in total relative abundance. In contrast, using water for reconstitution and storage can effectively limit nonspecific cleavages to 1%. Interestingly, the performances of different commercial trypsins were found to be quite distinct in their levels of nonspecific cleavages and responses to the two reconstitution conditions. Our findings demonstrate the importance of choosing the appropriate trypsin for tryptic digestion and the necessity of assessing the impact of trypsin reconstitution and storage on nonspecific cleavages. We advocate for manufacturers of commercial trypsins to reevaluate manufacturing processes and reconstitution/storage conditions to provide good cleavage specificity.
Topics: Acids; Humans; Peptide Fragments; Proteins; Proteolysis; Proteomics; Trypsin
PubMed: 32722706
DOI: 10.1371/journal.pone.0236740 -
Fish Physiology and Biochemistry Sep 2010Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200,...
Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS-PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N ( alpha )-p-tosyl-L: -lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60 degrees C, and the trypsin was stable below 50 degrees C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.
Topics: Amino Acid Sequence; Animals; Chromatography; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Intestine, Small; Japan; Molecular Sequence Data; Oncorhynchus; Phenylmethylsulfonyl Fluoride; Phylogeny; Sequence Analysis, Protein; Temperature; Trypsin; Trypsin Inhibitors
PubMed: 19680768
DOI: 10.1007/s10695-009-9336-4 -
Se Pu = Chinese Journal of... Mar 2022Protein complexes are involved in a variety of biological activities. Accurate and comprehensive characterization of the structures and interactions of protein complexes...
Protein complexes are involved in a variety of biological activities. Accurate and comprehensive characterization of the structures and interactions of protein complexes is crucial in determining their biological functions. Chemical cross-linking coupled with mass spectrometry (CXMS) is an emerging investigative technique for protein complexes. CXMS enables the sensitive high-throughput analysis of protein complexes without the requirements of molecular weight and purification. These attributes have spurred the increased use of CXMS for the structure and interaction characterization of purified protein complexes and complicated cell lysate samples. CXMS utilizes chemical cross-linking reagents to covalently connect two reactive amino acids in or between proteins that are spatially close to each other. Subsequently, the cross-linked proteins are digested into cross-linked peptides, followed by LC-MS/MS analysis, as well as database searching to provide cross-linking information for the composition, interaction, and structural site distance restrictions of protein complex identification. Therefore, identification of cross-linked sites has a decisive influence on the characterization of protein complexes. This identification is limited by the unsatisfactory quality of the cross-linked peptide spectrum. Insufficient b/y fragment ions and poor continuity of amino acid sequence matching lead to low coverage and accuracy of cross-linked site identification. Based on the complementary feature of mirror-cutting digestion, an orthogonal digestion strategy based on LysargiNase combined with trypsin was developed in this study. Trypsin is the most commonly utilized digestion enzyme in proteomics, with extremely high enzyme activity and specificity. Trypsin generates C-terminally charged peptides after lysine (K) and arginine (R). LysargiNase is a mirror protease complementary to trypsin that cleaves before the K and R residues. This generates peptides with an N-terminal positively-charged residue. Owing to the different physical and chemical micro-environments of the cross-linked peptides digested by LysargiNase and trypsin, the behavior of their detection ability in MS analysis is diverse. Using the orthogonal digestion strategy, both simple and complicated cross-linked samples were analyzed in this study. For the analysis of bovine serum albumin (BSA), 291 pairs of non-redundant cross-linked sites were obtained, of which 216 pairs of cross-linked sites were provided by trypsin digestion, whereas 75 pairs of cross-linked sites were exclusively supplied by LysargiNase digestion. Except for the 35% increase in the number of identified cross-linked sites, 32% of the spectra of the commonly identified cross-linked peptides have better quality with more b-type fragment ions and consecutive sequence matching. Furthermore, for the Escherichia coli sample, 726 pairs of cross-linked sites were obtained in total, among which, 624 and 274 pairs were identified from trypsin and LysargiNase digestion, respectively. LysargiNase digestion yielded 120 individual cross-linked sites, which resulted in a 16% increase in single trypsin digestion. Consistent with the BSA sample, the quality was improved in 35% of the spectra of commonly identified cross-linked peptides. Corresponding to the identified cross-linked peptides, 242 structural constraints with 607 pairs of intra-cross-linked sites and 29 sets of protein-protein interactions with 119 pairs of inter-cross-linked sites were obtained. The collective results demonstrated that, mirror cutting-assisted orthogonal digestion strategy could significantly increase the number of identified fragment ions and amino acid sequences matching the continuity of the spectra by contributing b-and y-type ions, respectively. This improved the accuracy and coverage of cross-linked peptide identification. The findings additionally demonstrate the superiority of our method in the accurate identification of the cross-linked peptide spectra and the increased number of identified cross-linked sites. In a word, this method is expected to provide new insights for the large-scale and highly accurate characterization of protein complexes.
Topics: Chromatography, Liquid; Digestion; Proteomics; Tandem Mass Spectrometry; Trypsin
PubMed: 35243832
DOI: 10.3724/SP.J.1123.2021.06010 -
PloS One 2017PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation of which has been implicated in promoting tumor progression. To date there are no...
PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation of which has been implicated in promoting tumor progression. To date there are no mesotrypsin-selective pharmacological inhibitors which could serve as tools for deciphering the pathological role of this enzyme, and could potentially form the basis for novel therapeutic strategies targeting mesotrypsin. A virtual screen of the Natural Product Database (NPD) and Food and Drug Administration (FDA) approved Drug Database was conducted by high-throughput molecular docking utilizing crystal structures of mesotrypsin. Twelve high-scoring compounds were selected for testing based on lowest free energy docking scores, interaction with key mesotrypsin active site residues, and commercial availability. Diminazene (CID22956468), along with two similar compounds presenting the bis-benzamidine substructure, was validated as a competitive inhibitor of mesotrypsin and other human trypsin isoforms. Diminazene is the most potent small molecule inhibitor of mesotrypsin reported to date with an inhibitory constant (Ki) of 3.6±0.3 μM. Diminazene was subsequently co-crystalized with mesotrypsin and the crystal structure was solved and refined to 1.25 Å resolution. This high resolution crystal structure can now offer a foundation for structure-guided efforts to develop novel and potentially more selective mesotrypsin inhibitors based on similar molecular substructures.
Topics: Amino Acid Sequence; Catalytic Domain; Databases, Pharmaceutical; Diminazene; Dose-Response Relationship, Drug; Drug Discovery; Escherichia coli; Humans; Hydrogen Bonding; Molecular Docking Simulation; Molecular Structure; Static Electricity; Trypsin; Trypsin Inhibitors; United States; United States Food and Drug Administration
PubMed: 28463992
DOI: 10.1371/journal.pone.0176694