Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In addition to determination of total selenium in nuts, the element distribution among different fractions (lipid extract, low molecular weight, and protein fractions), and speciation analysis were studied. Improved precision for total selenium determination was observed after elimination of lipids. Because selenium was not detected in any of the lipid extracts obtained from the different types of nuts (ICP-MS), in each determination and/or speciation procedure used in this work lipids were extracted (chloroform-methanol, 2:1) and discarded before analysis. In agreement with previously reported data, high selenium levels were found in Brazil nuts (those purchased without shells contained approximately a quarter the content than those purchased with shells) and significantly lower levels in walnuts, cashews, and pecans nuts. Low-molecular-weight compounds were extracted with perchloric acid (0.4 mol L(-1)) to furnish a fraction containing 3 to 15% of the total selenium in different types of nuts. The proteins were isolated from nut samples by dissolution in 0.1 mol L(-1) sodium hydroxide and subsequent precipitation with acetone. They were then dissolved in phosphate buffer pH 7.5. Analysis of protein fractions focused on selenium in two possible states - weakly and firmly bound to proteins. Results obtained for Brazil nuts by size-exclusion chromatography with on-line ICP-MS detection, in the absence and in the presence of beta-mercaptoethanol, showed that approximately 12% of total selenium was weakly bound to proteins. To obtain information about firmly bound selenium, the protein extracts were hydrolyzed enzymatically with proteinase K. Speciation was performed by means of ion-pairing HPLC-ICP-MS. The primary species found in all types of nuts was Se-methionine (19-25% of total selenium for different types of nuts).
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PMID:HPLC-ICP-MS determination of selenium distribution and speciation in different types of nut. 1217 80

In this work, the use of methanesulfonic acid for protein hydrolysis is proposed for evaluation of Se-methionine in yeast, Brazil nuts, and possibly other selenium-rich biological samples. The hydrolysis was carried out by heating the sample with 4 mol L(-1) acid at reflux for 8 h. Two chromatographic techniques (size-exclusion and ion-pairing) coupled with ICP-MS detection were used to compare the release of Se-methionine from proteins by enzymatic (proteinase K, protease XIV) and acid hydrolyses. A more efficient liberation of Se-methionine was observed by acid hydrolysis. For quantification, the sample extracts were introduced onto a C8 Alltima column, and the separation was achieved with a mobile phase containing 5 mmol L(-1) hexanesulfonic acid in citrate buffer (pH 4.5)/methanol (95:5). The results obtained by standard addition showed 816+/-17 micro g g(-1) and 36.2+/-1.5 micro g g(-1) of selenium in the form of Se-methionine in yeast and nuts, respectively (65% and 75% of total selenium).
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PMID:Hydrolysis of proteins with methanesulfonic acid for improved HPLC-ICP-MS determination of seleno-methionine in yeast and nuts. 1252 Apr 49

In this study, selenium species from Se containing proteins in mushrooms (Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1M NaOH, 30 mM Tris-HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris-HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample.
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PMID:Selenium speciation in Agaricus bisporus and Lentinula edodes mushroom proteins using multi-dimensional chromatography coupled to inductively coupled plasma mass spectrometry. 1623

In this work, size exclusion chromatography (SEC) with UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used to study the association of selenium to proteins present in Brazil nuts (Bertholletia excelsa) under five different extraction conditions. As expected, better solubilization of proteins was observed using 0.05molL(-1) sodium hydroxide and 1% sodium dodecylsulfate (SDS) in Tris/HCl buffer (0.05molL(-1), pH 8) as compared to 0.05molL(-1) HCl, 0.05molL(-1) Tris/HCl or hot water (60 degrees C). Due to non-destructive character of Tris-SDS treatment, this was applied for studying molecular weight (MW) distribution patterns of selenium-containing nut proteins. Three different SEC columns were used for obtaining complete MW distribution of selenium: Superdex 75, Superdex Peptide, and Superdex 200 were tested with 50mmolL(-1) Tris buffer (pH 8), 150mmolL(-1) ammonium bicarbonate buffer (pH 7.8), phosphate (pH 7.5), and CAPS (pH 10.0) mobile phases. Using Superdex 200 column, the elution of at least three MW fractions was observed with UV detection (200-10kDa) and ICP-MS chromatogram showed the co-elution of selenium with the two earlier fractions. The apparent MWs of these selenium-containing fractions were respectively about 107 and 50kDa, as evaluated from the column calibration. For further characterization of individual selenium species, the defatted nuts were hydrolyzed with proteinase K and analyzed by capillary electrophoresis (CE) with ICP-MS detection. The suitability of CE for the separation of selenite, selenate, selenocystine and selenomethionine in the presence of the nut sample matrix is demonstrated. Complete separation of the above mentioned selenium species was obtained within a migration time of 7min. In the analysis of nut extracts with CE-ICP-MS, selenium was found to be present mainly as selenomethionine.
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PMID:Studying the distribution pattern of selenium in nut proteins with information obtained from SEC-UV-ICP-MS and CE-ICP-MS. 1896 75

An analytical method for selenium species of selenate (SeVI), selenite (SeIV), selenomethionine (SeMet), selenocystine (SeCys2 ) and selenoethionine (SeEt) was established using high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). A Hamilton PRP X-100 reversed-phase anion exchange column (250 mm x 4.6 mm, 5 microm) with a 5 mmol/L citric acid buffer solution (pH 4.5, adjusted with 20% (v/ v) ammonia) as mobile phase was used for separation, and ICP-MS was used for detection. The five species were completely separated within 21 min. All the linear correlation coefficients of the five selenium species were greater than 0.999 5, and the detection limits of SeVI, SeIV, SeMet, SeCys2, SeEt were 0.4, 0.4, 5.6, 0.9 and 1.2 microg/L, respectively. The extraction procedure was studied for fresh mushroom and pork samples. For water-soluble selenium compounds, citric acid was a good extraction solution, and the recoveries were around 100% for inorganic selenium and in the range of 85.0% - 95.3% for SeMet; but worse for SeCys2 and SeEt. As for the proteinase K, the recoveries of SeCys2 and SeEt were raised to the range of 79.9% -91.5%. The method has the advantages of simple operation and good accuracy, and can be used for the quantitative determination of the five selenium species in food.
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PMID:[Determination of selenium species in food by high performance liquid chromatography with inductively coupled plasma mass spectrometry]. 2165 51

The prevalent use of engineered nanoparticles (ENPs) has increased our exposure to these particles. The current available analytical techniques fail to simultaneously quantify and analyze the physical properties of ENPs in biological tissues. Therefore, new methods are required to evaluate the exposure conditions to ENPs. Single particle inductively coupled plasma-mass spectrometry (sp-ICP-MS) is an attractive approach that can perform quantitative and qualitative analyses of ENPs. However, the application of this approach for biological samples is limited because of the lack of pretreatment methods for effectively recovering ENPs from biological tissues. In this study, we evaluated various pretreatment methods and identified the optimal pretreatment conditions for sp-ICP-MS analyses of ENPs in biological tissues using silver nanoparticles (nAg) as a model. We screened five reagents as pretreatment solvents (sodium hydroxide, tetramethylammonium hydroxide, nitric acid, hydrochloric acid, and proteinase K). Our results showed that treatment with sodium hydroxide was optimal for detecting nAg in the mouse liver. Moreover, this pretreatment method can be applied to other organs, such as the heart, lung, spleen, and kidney. Finally, we evaluated the applicability of this method by analyzing the quantity and physical properties of silver in the mouse blood and liver, after intravenous administration of nAg or silver ion. Our sp-ICP-MS method revealed that nAg administered into the blood was partially ionized and tended to be distributed in the particle form (approximately 80%) in the liver and in ionic form (approximately 95%) in the blood. In conclusion, we optimized pretreatment strategies for sp-ICP-MS evaluation of ENPs in biological tissues and demonstrated its applicability by evaluating the changes in the physical properties of nAg in the liver and blood. We also showed that partial changes from the particle form to the ionic form of nAg influences their kinetics and distribution when administered to mice.
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PMID:Optimization and Evaluation of Pretreatment Method for sp-ICP-MS to Reveal the Distribution of Silver Nanoparticles in the Body. 3113 47