Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C1389183 (autodigestion)
 317 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Capillary electrophoresis (CE) and matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated as alternatives to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis for peptide mapping with Staphylococcus aureus protease (V8) of a hydrophobic recombinant hepatitis C virus antigen, HC-31, which required 0.1% SDS for solubility. Controls (V8 only) or HC-31 digests were extracted with chloroform-methanol-water (1:4:3) to remove SDS, which interferes with MALDI-TOF, and high salt content, which affects CE. In two different runs by CE, the elution times of each of 11 peptide peaks were very reproducible (R.S.D. < 0.016). 25 fragments were resolved by MALDI-TOF-MS, including six smaller peptides (M(r) < 13 000) resulting from V8 autodigestion. MALDI-TOF-MS indicated that partial cleavages occurred, primarily at sites where there are paired glutamic and/or aspartic acid residues.
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PMID:Comparative peptide mapping of a hepatitis C viral recombinant protein by capillary electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry. 884 66

Enzymatic digestion of proteins is a key step in protein identification by mass spectrometry (MS). Traditional solution-based protein digestion methods require long incubation times and are limitations for high throughput proteomics research. Recently, solid phase digestion (e.g. trypsin immobilization on solid supports) has become a useful strategy to accelerate the speed of protein digestion and eliminate autodigestion by immobilizing and isolating the enzyme moieties on solid supports. Monolithic media is an attractive support for immobilization of enzymes due to its unique properties that include fast mass transfer, stability in most solvents, and versatility of functional groups on the surfaces of monoliths. We prepared immobilized trypsin monolithic capillaries for on-column protein digestion, analyzed the digested peptides through LC/FTICR tandem MS, and compared peptide mass fingerprinting by MALDI-TOF-MS. To further improve the digestion efficiency for low abundance proteins, we introduced C4 functional groups onto the monolith surfaces to combine on-column protein enrichment and digestion. Compared with immobilized trypsin monolithic capillaries without C4, the immobilized trypsin-C4 monolith showed improved digestion efficiency. A mechanism for increased efficiency from the combination of sample enrichment and on-column digestion is also proposed in this paper. Moreover, we investigated the effects of organic solvent on digestion and detection by comparing the observed digested peptide sequences. Our data demonstrated that all columns showed good tolerance to organic solvents and maintained reproducible enzymatic activity for at least 30 days.
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PMID:A bifunctional monolithic column for combined protein preconcentration and digestion for high throughput proteomics research. 1715 Apr 20

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.
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PMID:A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties. 1822 Mar 46