Samples proteolytic enzymes

Factors to be considered in maldng the selection of chromatography processing steps are cost, sample volume, protein concentration and sample viscosity, degree of purity of protein product, presence of nucleic acids, pyrogens, and proteolytic enzymes. Ease with which different types of adsorbents can be washed free from adsorbed contaminants and denatured proteins must also be considered. 

A typical procedure is shown in Figure 2. Other dyes besides ethidium can be used, although ethidium has an advantage in that its excitation emission bands are well removed from any protein absorbances. A standard curve can be constructed for the nucleic acid of concern and the limits of detection established. In Step 3, proteolytic enzymes may be substituted for heparin, or the step may be bypassed in the case of proteins which do not interfere. After measurement of the unknown sample the nucleic acid concentration may be simply calculated or read from the standard curve. 

The tissue or cell sample is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS), which disrupts the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acids and so leaves the single nucleotides from RNA digestion in the aqueous layer. 

Blood, urine, and tissues Addition of sample to internal standard addition of proteolytic enzyme equilibration at elevated temperature analysis of headspace gas GC/ECD At least 1 ppm No data Streete et al. 1992... 

Glucagon is extensively degraded in the liver and kidney as well as in plasma and at its tissue receptor sites. Because of its rapid inactivation by plasma, chilling of the collecting tubes and addition of inhibitors of proteolytic enzymes are necessary when samples of blood are collected for immunoassay of circulating glucagon. Its half-life in plasma is between 3 and 6 minutes, which is similar to that of insulin. 

Semisolid samples such as muscle and liver tissues can be homogenized by blending with water or an appropriate aqueous solution such as a buffer in a mechanical or an ultrasonic device to expose the residue to the extraction solvent. Fatty tissue samples are sometimes subjected to heating at 40 or 60 C until fat becomes liquid, prior to extraction of the analytes with hexane (433) or acetonitrile (434), respectively. An alternative pretreatment approach is the enzymatic digestion of the tissue by means of proteolytic enzymes such as subtilisin A (429, 435-437). 

Figure 5 also shows two 10-hr samples, 10a and 10b. Sample 10a was stored in solution at 4°C for one week, while sample 10b was stored frozen and then thawed immediately before application to the polyacrylamide gel. Both samples show the same protein band pattern. If proteolytic enzymes in the culture filtrate had acted on and partially degraded the extracellular proteins, a different band pattern would have been expected. Thus no product-precursor relationship appeared to exist between the various extracellular proteins in a culture filtrate of Thermoactinomyces. Moreover, it seems as if this organism produces at least three different extracellular cellulolytic enzymes simultaneously. 

Four different extraction procedures have been evaluated for a quantitative recovery of Se species during extraction from cod muscle. The highest Se recoveries were obtained in the presence of enzymes, whereas only 5 percent of total Se in cod was extracted when a soft extraction procedure (MeOHDHCl) was used [42]. The use of proteolytic enzymes allowed to extract more than 90 percent of Se present in the wheat Bour samples and more than 85 percent of Se from yeast samples [43]. 

All of the three phases of enzymatic methods were dominated by proteolytic enzymes. However, nonproteolytic ones were also addressed for special purposes to indirectly assist proteolysis, for example, by providing access to proteins to be hydrolyzed through the enzymatic decomposition of the matrix constituents. At the same time, the use of nonproteolytic enzymes might be promoted in the future to study the possibly nonprotein-associated Se macromolecules which have already been observed in the case of numerous real samples, for example, edible mushroom [58, 80], but so far have never been investigated in detail. 

After the introduction of pronase E, other more or less nonsubstrate-specific proteolytic enzymes have been applied to assist Se speciation. Most of them were derived from DNA/RNA clean-up protocols. The new enzymes (subtilisin from Bacillus licheniformis, also named protease VIII, EC 3.4.21.14 proteinase K from Tritirachium album, EC 3.4.21.64 the crude Novo Nordisk product of Flavourzyme from Aspergillus oryzae) proved to be capable of extracting Se with varying yields and chromatographic recovery of Se species. It is important to highlight that the latter parameter also depends on the instrumentation available. In this regard, different recovery values for the same samples reported by independent research groups do not necessarily indicate successful or unsuccessful sample preparation. Similarly, extraction efficiency (defined as the ratio of extracted Se to total Se in the sample) cannot be used as such for comparison purposes because sample preparation may include some extra steps, for example, TCA precipitation or ultrafiltration, which may reduce this value even by 10-20 percent. 

The Use of Nonproteolytic Enzymes Prior to Se Speciation It goes without saying that food samples contain components other than proteins. This fact can be related to the observation that some sample preparation attempts based on the sole use of proteolytic enzymes have failed, for example, protease XIV could not extract more than 8 percent of Se from Se-enriched lactic acid bacteria with a chromatographic recovery of 27 percent [77]. There are two possible explanations for this phenomenon (1) formerly unidentified and nonprotein-bound Se species might be present in the samples under test, which cannot be released by proteolytic enzymes from the matrix constituents under the experimental conditions adopted (2) the unextracted Se species are protein-bound, but the proteolytic attack is hampered by a matrix constituent, hindering any enzymatic access. 

A mixture of protease inhibitors, which is used to preserve protein integrity during the processing of samples for subsequent analysis. The term cocktail refers to a mixture of components. A protease inhibitor cocktail is composed of a broad spectrum of protease inhibitors and intends to inhibit the diverse proteolytic enzymes found in tissue extracts and biological fluids. See Pringle, J.R., Methods for avoiding proteolytic artifacts in studies with enzymes and other proteins from yeasts. Methods Cell Biol. 12, 149-184, 1975 Dmbin, D.G., Miller, K.G., and Botstein, D., Yeast... 

Ever since its inception in forensic toxicology [68], enzymatic digestion has been extensively used to isolate drugs from forensic samples. Carpenter [69] was the first to develop an analytical procedure in which subtilisin, a proteolytic enzyme, was used to determine Cd, Cu, Pb and Tl from human liver and kidney tissues. 

Mass spectrometry (MS) permits the identification of proteins by the determination of the exact mass of peptides, and the fragmentation of these peptides to determine the amino acid sequence. Protein samples, such as purified organelles or protein complexes are usually separated by their relative molecular weight on SDS-PAGE gels. The protein bands are visualized by dyes such as Coomassie brilliant blue and then are excised from the gel. The protein-containing gel slices are washed and incubated with proteolytic enzymes such as trypsin and the resultant peptides extracted from the gel-slices. 

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