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)

The resolution potential of reverse-phase high-performance liquid chromatography (HPLC) for peptide analysis of hydrophobic viral membranes has been investigated, using as model the membrane (M) protein of influenza virus. Proteolytic digests of 125I-labelled M protein and CNBr fragments, extracted from radioiodinated whole virus, have been separated on a uBondapak C18 column with an isopropanol or acetonitrile solvent system. Peptide mapping of trypsin digests of M protein from A/PR/8/34 (H1N1) and A/chicken/Germany/N/49 (H10N7) viruses was identical, whereas Staphylococcus aureus V8 protease digests showed minor differences in at least two peptides. The results also show that HPLC is a powerful tool for the separation of proteolytic digests of viral proteins, since the peptide maps are highly reproducible and recovery was greater than 85%.
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PMID:Peptide mapping of 125I-labelled membrane protein of influenza viruses by reverse-phase high-performance liquid chromatography. 707 81

A comprehensive approach for the structural microanalysis of collagen based of collagen based on high-performance liquid chromatography (h.p.l.c.) has been developed using calf skin type I collagen as a model system. The alpha, beta and gamma components were separated, after heat denaturation, on a TSK 4000 SW gel permeation column, using a nonvolatile buffer. Monitoring at 210 nm permits the detection of 1 microgram of a single chain. The alpha 1(I) and alpha 2(I) chains were completely resolved using a large-pore reversed-phase column (Vydac 201 TP 4.6) eluted by an aqueous acetonitrile gradient (24-48%) containing 0.01 M heptafluorabutyric acid as an ion-pairing agent. The purified alpha 1(I) chain was digested with CNBr and the resulting fragments separated in the same chromatography system with a gradient containing a 12.8-44.8% acetonitrile gradient. The purified alpha 1(I)CB 3 peptide was further cleaved with trypsin and the resulting peptides separated first by a similar chromatography with a 4-32% acetonitrile gradient. Resolution of some poorly separated peptides was obtained by a rechromatography using trifluoroacetic acid as counterion. The isolated peptides were hydrolyzed and identified by their amino-acid composition. Sequencing of h.p.l.c.-purified alpha 1(I)CB 3 was also performed to demonstrate the suitability of the technique for the preparation of peptides for amino-acid sequencing. This study demonstrates that detailed structural analysis can be performed on 3 mg of a purified collagen.
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PMID:A comprehensive approach to the study of collagen primary structure based on high-performance liquid chromatography. 711 47

The effect of the venom of the spider Lycosa erythrognatha on the frog sciatic nerve was investigated with the single sucrose-gap method. Solutions containing the crude venom (40 micrograms protein/ml) markedly increased the duration of compound action potentials and caused the appearance of long-lasting depolarizing post-potentials. These effects were only partially (20%) reversed by extensive washing with control solution. The active material was sensitive to proteolytic treatments with pronase or trypsin and was separated with 20% acetonitrile and 0.1% trifluoroacetic acid by reverse phase chromatography. The effect of this fraction (LycIV) on the post-potential amplitude was concentration-dependent, and was fitted with a quadratic hyperbola having a half maximal effect of 0.9 microgram protein/ml. SDS-polyacrylamide gel electrophoresis of LycIV showed an enriched polypeptide band with apparent molecular weight of approximately 8 kDa. The observed effects were similar to those of toxins that inhibit sodium channel inactivation and different from the effects of potassium channel blockers. Pore formation or membrane disruption could be ruled out. It was concluded that the venom contains a neurotoxic polypeptide that alters the repolarization of action potentials, probably by inhibiting sodium channel inactivation.
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PMID:Partial purification and pharmacological characterization of a neurotoxic fraction isolated from the venom of the spider Lycosa erythrognatha. 754 89

Bovine erythrocyte acetylcholinesterase was prepared for tryptic digestion by radiomethylating with [14C]HCHO and NaCNBH3, cleaving with purified bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the glycoinositol phospholipid anchor, and reducing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with trypsin. In the conventional procedure, acetylcholinesterase was treated with 6 M guanidine hydrochloride for 40 min at room temperature and dialyzed. In a new procedure, acetonitrile (CH3CN) was added to 30% v/v for 10-15 min at room temperature and then removed by vacuum evaporation. The CH3CN concentration during evaporation could be estimated from the apparent pH of the solution (20 mM phosphate buffer), which varied linearly over the range of 0-75% CH3CN. CH3CN was removed in a mixture of constant composition (approximately 11% H2O-89% CH3CN), so that a final CH3CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded comparable HPLC profiles for A215 and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gentle conditions.
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PMID:Protein denaturation by addition and removal of acetonitrile: application to tryptic digestion of acetylcholinesterase. 771 Jan 3

An improved and simplified procedure for enzymatic digestion of proteins bound to polyvinylidene difluoride (PVDF) membranes for obtaining internal protein sequence data is presented. This improved procedure is compatible with various enzymes (trypsin, endoproteinase Lys-C, endoproteinase Glu-C, and clostripain) and is performed in the presence of 1% hydrogenated Triton X-100 (RTX-100)/10% acetonitrile/100 mM Tris, pH 8.0, followed by microbore HPLC purification of the recovered peptides. Previously published techniques required treatment of the PVDF-bound protein with polyvinylpyrrolidine M(r) 40,000 (PVP-40) prior to digestion, in order to prevent adsorption of the enzyme to the membrane. Unfortunately, contaminants produced from residual PVP-40 interfere with subsequent peptide mapping. We have found that when RTX-100 is used in the digestion buffer, no pretreatment of the PVDF-bound protein with PVP-40 is necessary. Advantages of this improved (one-step) procedure over the two-step PVP-40 procedure are (a) the elimination of undesirable contaminants associated with PVP-40, (b) a decrease in the time required for the technique, and (c) a reduction in sample manipulation. Peptide maps and recoveries from PVDF-bound standard proteins (4 micrograms each) enzymatically digested with this one-step method are compared with those obtained from the standard PVP-40 method. In addition, peptide maps and internal sequence data from low-level quantities of unknown proteins enzymatically digested with the improved procedure are presented. To date, this improved, one-step procedure has been successfully applied to 52 PVDF-bound unknown proteins (0.7-10 micrograms) of varying molecular weight (19-300 kDa) for which internal sequence data were obtained.
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PMID:An improved procedure for enzymatic digestion of polyvinylidene difluoride-bound proteins for internal sequence analysis. 805 43

Confirmation of a protein's cysteine content and of its location within the amino acid sequence is crucial in investigating the structural integrity of recombinant proteins. A combination of thiol-specific chemistry and peptide mapping by reversed-phase microbore HPLC was used to confirm the presence and map the location of cysteine residues in the primary sequences of recombinant porcine growth hormone and human tumor necrosis factor alpha. Recombinant proteins were conjugated with a hydrophobic iodoacetamide derivative, dimethylaminoazo-benzene iodoacetamide, and digested with trypsin. The peptide fragments were separated on a C8 microbore reversed-phase column using a linear acetonitrile gradient. The peptides containing the cysteine residues were selectively identified by monitoring with a diode-array detector at 215 nm with the reference wavelength set at 450 nm. Cysteine-containing peptides could be readily distinguished as inverted "negative" peaks relative to the baseline and noncysteine-containing peptides. Isolated peptide fragments were then sequenced in order to confirm the location of the cysteines in the proteins. This approach offers the benefits of selectively and rapidly identifying, from a single chromatrophic step, the cysteine-containing peptides of proteins. Furthermore, the use of the labeling reagent renders the cysteine-containing peptides more hydrophobic, thereby making them easier to separate from noncysteine-containing peptides.
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PMID:Identification of cysteine-containing peptides during the peptide mapping of recombinant proteins. 825

A precolumn fluorescence derivatization method combined with high-performance liquid chromatography is described for the sensitive and selective determination of N-terminal tryptophan-containing peptides. The peptides and tryptophan were converted into fluorescent derivatives with glyoxal in a moderately acidic medium (pH 4.5). The derivatives were separated on a reversed-phase column with isocratic elution with an aqueous mobile phase composed of acetonitrile, methanol and phosphate buffer (pH 6.0), and subsequently detected by fluorimetry. The derivatization technique provided the respective N-terminal tryptophan-containing oligopeptides with single fluorescent peaks in chromatography. The detection limits for the peptides were 55-382 fmol per 100-microliters injection volume at a signal-to-noise ratio of 3. The method also allowed the facile detection of an N-terminal tryptophyl fragment in the enzyme reaction mixture of dynorphin A with trypsin.
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PMID:High-performance liquid chromatography of N-terminal tryptophan-containing peptides with precolumn fluorescence derivatization with glyoxal. 826 56

The photoreactive ADP analogue 8-N3-ADP binds in the dark to the catalytic site of the sarcoplasmic reticulum Ca-ATPase. An apparent Kd value of 30 microM has been deduced from competition with ADP in the presence of EGTA. Photoirradiation of Ca-ATPase with 8-N3-[3H]ADP in the presence of calcium results in irreversible inhibition of ATPase activity with corresponding stoichiometries of covalently and specifically photolabeled Ca-ATPase. The site of photolabeling of the Ca-ATPase in the presence of calcium has been explored. Controlled trypsin digestion of the labeled protein shows that 8-azido-ADP is incorporated in the B subfragment. Extensive trypsin digestion of the labeled protein releases a small peptide as revealed by gel filtration chromatography (Sephadex G-50). Further HPLC purification on a reverse-phase column (C8) eluted with a water/acetonitrile gradient buffered at pH 6 or at pH 2 gives a single labeled peptide. Edman degradation of that isolated peptide, as well as the amino acid composition, shows that it contains five amino acid residues (Val-530-Arg-534) with the radioactivity localized on Thr-532 and Thr-533.
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PMID:Identification of amino acid residues photolabeled with 8-azidoadenosine 5'-diphosphate in the catalytic site of sarcoplasmic reticulum Ca-ATPase. 838 81

The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their L-form or D-form. Like Asn-Gly, the sequence Asp-Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128-134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where L-Asp was replaced by D-Asp, L-isoAsp, D-isoAsp or the L-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was D-Asp, L-Asp, L-isoAsp, D-isoAsp and L-cyclic imide. Reverse-phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was D-isoasp, L-isoASp, L-Asp, D-Asp and L-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only L-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.
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PMID:Synthesis of stereoisomers and isoforms of a tryptic heptapeptide fragment of human growth hormone and analysis by reverse-phase HPLC and capillary electrophoresis. 863 46

A rapid and simple method for analyzing cathepsin D in breast tissue based on capillary zone electrophoresis (CZE) is described. After incubating the tissue extracts with hemoglobin as a substrate, a specific peptide is cleaved and separated by CZE in less than 5 min. This peptide is not produced by the action of pepsin or trypsin. It is inhibited by the addition of pepstatin, a specific inhibitor for cathepsin D. Human hemoglobin acted as a better substrate than bovine hemoglobin. The test compared well to a radioimmunoassay. We have shown that peptides can be stacked by the use of acetonitrile. The method demonstrates the advantages of CZE for assay of proteolytic enzymes in general.
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PMID:Analysis of cathepsin D from breast tissues by capillary electrophoresis. 887 48


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