Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper deals with the interactions of chlorogenic, caffeic, and quinic acids and p-quinone with myoglobin. The myoglobin derivatives formed have been characterized in terms of physicochemical properties and susceptibility to proteolysis. The results show that the free amino group and tryptophan contents of the myoglobin-phenol derivatives decrease with the increasing extent to which the protein becomes derivatized. Furthermore, the solubility of myoglobin-phenol derivatives decreases in the pH range 3.5-6.5 as compared to solubility of the native protein. The reaction also influences the hydrophilic-hydrophobic character of the protein. The isoelectric point of the derivatized myoglobin is shifted to a lower pH value, and formation of high molecular fractions is also documented. This paper also demonstrates the influence of the protein derivatization with plant phenols on susceptibility to digestion by trypsin, alpha-chymotrypsin, and pepsin, determined in vitro. The enzymatic digestion of the derivatized proteins is adversely affected.
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PMID:Physicochemical properties and susceptibility to proteolytic digestion of myoglobin-phenol derivatives. 1082 62

The use of thermal denaturation of proteins prior to in-solution digestion and mass spectral peptide mass mapping is reported. Thermal denaturation is preferred over chemical denaturation because it does not require purification/concentration prior to mass spectral analysis. Enzymatic digestions of proteins that are resistant to proteolysis are significantly enhanced by thermal denaturation. Native proteins that are sensitive to proteolysis show similar or slightly lower digestion yields following thermal denaturation. Proteins that are resistant to digestion become more susceptible to digestion, independent of protein size, following thermal denaturation. For example, amino acid sequence coverage from digest fragments increases from 15 to 86% in myoglobin and from 0 to 43% in ovalbumin. This leads to more rapid and reliable protein identification by MALDI peptide mass mapping. Although some proteins aggregate upon thermal denaturation, the protein aggregates are easily digested by trypsin and generate sufficient numbers of digest fragments for protein identification.
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PMID:Thermal denaturation: a useful technique in peptide mass mapping. 1085 53

A simple theoretical model is proposed for evaluation of the optical titration behaviour of tyrosyl and carboxyl residues in proteins. The pK values involved in the model are computed using the semi-empirical method. The titration curves are calculated using the values of the molar absorption differences for tyrosyl residues in the ultraviolet (UV) region at 245 and 295 nm, and for carboxyl residues in the infrared (IR) region at 1565 and 1707 cm(-1), respectively. The theoretical tyrosyl titration curves are compared with the experimental data for lysozyme, myoglobin and chymotrypsinogen (available in the literature). This approach provides a good tool for distinguishing between the ionisation and the conformational changes in the alkaline range. The quantitative evaluation of the change of molar extinction coefficients as a function of pH in the case of carboxyl titration for lysozyme, trypsin and cytochrome c shows a good agreement with the experimental titration data.
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PMID:Spectrophotometric titration of ionisable groups in proteins: a theoretical study. 1098 94

The trypsin digest of a mixture of two proteins, namely cytochrome c and myoglobin, was first separated in the first dimension by high-performance liquid chromatography (HPLC). Fractions from the HPLC were collected every 30s with the aid of a fraction collector into a 96-well microtiter plate. After concentration, all the collected fractions were analyzed simultaneaosly in the second dimension by a 96-array capillary electrophoresis system. The labeled peptides were detected by laser-induced fluorescence. An internal standard, allura red, was added to all the fractions, prior to capillary electrophoretic analysis. The internal standard serves two functions, migration time correction and signal intensity correction. The data are presented in two different formats, as an electropherogram of all the fractions and in a two-dimensional (2-D) format. The 2-D plot of the data shows the density of each spot, which corresponds to the concentration of the migrating peptides. The total experimental time for the HPLC and capillary electrophoretic analyses ist less than 1 h, which ist much faster than using 2-D slab-gel electrophoresis or single-capillary capillary electrophoresis.
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PMID:Multidimensional high performance liquid chromatography--capillary electrophoresis separation of a protein digest: an update. 1135 38

The rate of protein digestion imposes significant limitations on high-throughput protein identification using mass spectrometry. In this report, we demonstrate that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile. The rates of protein digestion in organic solvents, as indicated by the abundances of digest fragment ions in the mass spectrum, are increased relative to aqueous solution. In addition, amino acid coverage for the analyzed proteins increases in the presence of the organic solvents, and proteins that are resistant to proteolysis are readily digested. For example, a 68% amino acid sequence coverage was attained from a tryptic digest of myoglobin in < 5 min from an 80% acetonitrile solution, whereas no digest fragments were detected from a 5 min digestion in an aqueous solution. Moreover, the tryptic digestion of a complex protein mixture in an organic-aqueous solvent system showed significantly enhanced digestion for nearly all of the protein components. Enzymatic digestion in an organic-aqueous solvent system is a rapid, simple, and effective peptide mass-mapping technique.
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PMID:Proteolysis in mixed organic-aqueous solvent systems: applications for peptide mass mapping using mass spectrometry. 1140 17

The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the (1)H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The (13)C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH(2) group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system.
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PMID:Oxidative modification of tryptophan 43 in the heme vicinity of the F43W/H64L myoglobin mutant. 1148 19

Changes in the absorbance spectrum of tetraphenylporphyrin sulfonate (TPPS) are observed that are unique for the proteins lysozyme, luciferase, apomyoglobin, myoglobin, gamma globulin, insulin, RNAase, phosphotransacetylase, papain, ovalbumin, bovine serum albumin (BSA), protamine sulfate, and polylysine. The absorbance spectrum of porphyrins is different for native compared with heat denatured RNAase. A unique absorbance wavelength red shift is observed with trypsin when trypsin inhibitor is present, indicating that porphyrins incorporated with proteins can detect conformational changes in the protein. The absorbance spectrum of the Soret band of TPPS undergoes bathochromic shifts upon addition of local anesthetics to acetylcholine esterase (AChE), suggesting that the absorbance spectrum of porphyrins can be used as a reporter of the presence of inhibitors of AChE by indicating conformational changes on binding of the inhibitor.
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PMID:Spectroscopic determination of acetylcholine esterase-inhibitor complex: determination of conformational shifts of proteins. 1167 86

The interaction of trypsin-digested bovine cytochrome b(5) (cyt b(5)) with horse heart myoglobin (Mb) and the interprotein electron transfer (ET) between these redox partners have been studied to gain better understanding of ET processes between weakly bound protein partners. The bimolecular rate constant ( k(2)) for photo-induced ET between zinc-substituted Mb (ZnMb) and cyt b(5) decreases with increasing ionic strength, consistent with the predominantly electrostatic character of this complex. The formation of a protein-protein complex has been confirmed and the binding affinities of metMb and ZnMb for cyt b(5) have been measured by two techniques: (1)H NMR titrations at pH 6.0 give binding constants of K(a) approximately (1.0+/-0.1)x10(3) M(-1) for metMb and K(a) approximately (0.75+/-0.1)x10(3) M(-1) for ZnMb; isothermal calorimetry gives K(a) approximately (0.35+/-0.1)x10(3) M(-1) for ZnMb. Brownian dynamic (BD) simulations show that cyt b(5) binds over a broad surface of Mb that includes its heme edge. The experimental results are described in terms of a dynamic docking model which proposes that Mb binds cyt b(5) in a large ensemble of protein binding conformations, not one or a few dominant ones, but that only a small subset are ET reactive. Aided by the BD simulations, this model explains why k(2) decreases with increasing pH: increasing pH not only weakens the binding affinity but also reduces the number of binding conformations with high ET reactivity.
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PMID:Dynamic docking and electron transfer between myoglobin and cytochrome b(5). 1207 63

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.
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PMID:Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins. 1208 38

Enzymatic microreactors have been prepared in capillaries and on microfluidic chips by immobilizing trypsin on porous polymer monoliths consisting of 2-vinyl-4,4-dimethylazlactone, ethylene dimethacrylate, and acrylamide or 2-hydroxyethyl methacrylate. The azlactone functionalities react readily with amine and thiol groups of the enzyme to form stable covalent bonds. The optimized porous properties of the monoliths lead to very low back pressures enabling the use of simple mechanical pumping to carry out both the immobilization of the enzyme from its solution and the subsequent analyses of substrate solutions. The Michealis-Menten kinetic characteristics of the reactors were probed using a low molecular weight substrate: N-alpha-benzoyl-L-arginine ethyl ester. The effects of immobilization variables such as the concentration of trypsin in solution and percentage of azlactone functionalities in the monolith, as well as the effect of reaction time on the enzymatic activity, and of process variables such as substrate flow velocity and residence time in the reactor, were studied in detail. The proteolytic activity of the enzymatic microreactor on chip was demonstrated at different flow rates with the cleavage of fluorescently labeled casein used as a substrate. The excellent performance of the monolithic microreactor was also demonstrated with the digestion of myoglobin at the fast flow rate of 0.5 microL/min, which affords a residence time of only 11.7 s. The digest was then characterized using MALDI-TOF MS, and 102 out of 153 possible peptide fragments were identified giving a sequence coverage of 67%.
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PMID:Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. 1219 78


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