A trypsin and chymotrypsin inhibitor from the metacestodes of<i>Taenia pisiformis</i>

Németh, István; Juhász, Sándor

doi:10.1017/s0031182000000901

Cited by 13 publications

(7 citation statements)

References 22 publications

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“…These structural and morphological characteristics of CaCO 3 microparticles are directly determined by the mechanism of their formation from supersaturated solutions. It was found earlier (31)(32)(33)(34)37) that the growth of CaCO 3 microparticles is a kind of colloidal aggregation of primary CaCO 3 nanoparticles, instantaneously formed with mixing of calcium chloride and sodium carbonate solutions, into spherical micron-sized microparticles, which further slowly recrystallize in water into rhombohedral calcite crystals. Thus, to get the porous CaCO 3 microspheres, the recrystallization process should be stopped at the stage of their formation by filtering and thorough washing with water and then with acetone, followed by drying at air.…”

Section: Resultsmentioning

confidence: 99%

“…Protein concentration in solution was determined according to Bradford (35). The enzymatic activity of R-chymotrypsin was analyzed using BTEE as a substrate (24,37). The kinetics of hydrolysis was followed by the increase of the optical absorption at 253 nm of the reaction mixture obtained after rapid mixing in a 1-cm quartz cell of an aliquot of BTEE solution in methanol (0.2 mL, 0.3 mg/mL) with R-chymotrypsin solution (1.8 mL, 0.1-0.4 mg/mL) in 0.05 M Tris-HCl buffer, pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Protein-Calcium Carbonate Coprecipitation: A Tool for Protein Encapsulation

Петров

Volodkin

Sukhorukov

2008

Biotechnol Progress

365

352

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A new approach of encapsulation of proteins in polyelectrolyte microcapsules has been developed using porous calcium carbonate microparticles as microsupports for layer-by-layer (LbL) polyelectrolyte assembling. Two different ways were used to prepare protein-loaded CaCO3 microparticles: (i) physical adsorption--adsorption of proteins from the solutions onto preformed CaCO3 microparticles, and (ii) coprecipitation--protein capture by CaCO3 microparticles in the process of growth from the mixture of aqueous solutions of CaCl2 and Na2CO3. The latter was found to be about five times more effective than the former (approximately 100 vs approximately 20 mug of captured protein per 1 mg of CaCO3). The procedure is rather mild; the revealed enzymatic activity of alpha-chymotrypsin captured initially by CaCO3 particles during their growth and then recovered after particle dissolution in EDTA was found to be about 85% compared to the native enzyme. Core decomposition and removal after assembly of the required number of polyelectrolyte layers resulted in release of protein into the interior of polyelectrolyte microcapsules (PAH/PSS)5 thus excluding the encapsulated material from direct contact with the surrounding. The advantage of the suggested approach is the possibility to control easily the concentration of protein inside the microcapsules and to minimize the protein immobilization within the capsule walls. Moreover, it is rather universal and may be used for encapsulation of a wide range of macromolecular compounds and bioactive species.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Protein-Calcium Carbonate Coprecipitation: A Tool for Protein Encapsulation

Петров

Volodkin

Sukhorukov

2008

Biotechnol Progress

365

352

View full text Add to dashboard Cite

show abstract

“…), which demonstrated the specificity of the proposed method in the analysis of complex samples and indicated that interference from co‐eluted ions with quantitation results was negligible. The measurement accuracy for trypsin and chymotrypsin in the same samples was also examined by comparing the results with those obtained from BAEE and BTEE methods, which were also reliant on the substrate cleavage to assay protease activities and showed relative errors less than 5.2%. Thus, the proposed peptide‐encoding strategy could be potentially applicated in clinical diagnosis and expanded to protease activity assays in blood samples, while necessary dilution and desalting protocols might be needed to obtain sacrifatory results.…”

Section: Resultsmentioning

confidence: 99%

Peptide codes for multiple protease activity assay via high‐resolution mass spectrometric quantitation

Liu

Feng

et al. 2016

Rapid Comm Mass Spectrometry

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RATIONALE: Proteases, which are involved in a number of biological processes, play important roles in human health. Alterations in protease activities have been associated with many diseases. Thus, effective methods for multiple protease assay are very essential and may help to discover more about their functions in different physiological processes. Here, we proposed a strategy of peptide codes for label-free analysis of multiple protease activities using high-resolution mass spectrometry (HRMS). METHODS:The peptide codes were designed to contain a coding region and the substrate of the protease, respectively. Upon the cleavage of target proteases, the coding regions could be rapidly identified and directly quantitated by accurate m/z extractions without internal standard. Therefore, the coding region could be used as the unique "Protease ID" for the identification of the corresponding protease, and the amount of the cleavage product was used for protease activity analysis. RESULTS: The method was validated by using trypsin and chymotrypsin as model proteases, with the designed "Trypsin ID" and "Chymotrypsin ID" occurring at m/z 761.270 and 711.243, respectively. The peak area of "Protease ID" was proportional to trypsin and chymotrypsin concentration in the range from 0.01 to 10 nM and 10 to 2000 nM, respectively. The proposed method could also be used for screening of protease inhibitors, which might be of great importance for drug discovery. CONCLUSIONS: This method provided a label-free and accurate quantitative platform for convenient identification and activity analysis of multiple proteases, which could potentially be applied in clinical diagnosis.

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“…To assess the accuracy of the proposed method, the activities of trypsin and chymotrypsin were also detected with BAEE ( N -α-benzoyl- l -arginine-ethylester at 253 nm) and BTEE ( N -α-benzoyl- l -tyrosine-ethylester at 256 nm) methods, respectively. − Briefly, the assay was carried out in 0.1 M Tris-HCl buffer (pH 8.0) containing 6 mM CaCl 2 , 10 μM substrate (BAEE or BTEE), and trypsin or chymotrypsin at different concentrations, or 10× diluted samples. The absorbance was measured at 25 °C on a UV-3600 spectrophotometer (Shimadzu, Japan).…”

Section: Methodsmentioning

confidence: 99%

Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

Liu

2015

Anal. Chem.

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A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

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A trypsin and chymotrypsin inhibitor from the metacestodes ofTaenia pisiformis

Cited by 13 publications

References 22 publications

Protein-Calcium Carbonate Coprecipitation: A Tool for Protein Encapsulation

Protein-Calcium Carbonate Coprecipitation: A Tool for Protein Encapsulation

Peptide codes for multiple protease activity assay via high‐resolution mass spectrometric quantitation

Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

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