Phosphatase Test

This important test is done to confirm the absence of active alkaline phosphatase enzymes from pasteurised milk. If the active enzyme is still present, it will dephosphorylate added p-nitrophenyl phosphate and produce p-nitrophenyl whose yellow colour can be monitored. 

Some kinases are specific in action while others are broad spectrum and may assist in phosphorylation of several different proteins. The latter type, however, are often named after the protein upon which the enzyme was first found to act. Kinase-catalysed reactions are usually not reversible moreover it appears that different kinases may phosphorylate a given protein at different sites. 

The action of some protein kinases is stimulated by cyclic AMP and usually to a much less extent by c-GMP and other cyclic nucleotides (below). There have been attempts to classify kinases according to the type of protein on which they act (e.g. caseins or histones), or whether or not they are stimulated by c-AMP. 

Phosphatases, like kinases, can be specific or broad spectrum in their action. Phosphatases which dephosphorylate protein can usually act on other phosphomonoesters, however. There are phosphatases which act on caseins and those which act on histones, and so on. 

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Phosphatase Phosphatase enzymes Phosphatase tests Phosphatation Phosphate... 

Phosphatase Test. The phosphatase [9001-78-9] test is a chemical method for measuring the efficiency of pasteurization. AH raw milk contains phosphatase and the thermal resistance of this enzyme is greater than that of pathogens over the range of time and temperature of heat treatments recognized for proper pasteurization. Phosphatase tests are based on the principle that alkaline phosphatase is able, under proper conditions of temperature and pH, to Hberate phenol [108-95-2] from a disodium phenyl phosphate substrate. The amount of Hberated phenol, which is proportional to the amount of enzyme present, is determined by the reaction of Hberated phenol with 2,6-dichloroquinone chloroimide and colorimetric measurement of the indophenol blue formed. Under-pasteurization as well as contamination of a properly pasteurized product with raw milk can be detected by this test. 

Two methods commonly used to test for seminal stains are the acid phosphatase test and the Florence test. Both tests were developed on the basis of the reaction of an introduced compound with substances that are present in seminal fluid. Positive results for these tests are either the formation of a characteristic color or the formation of specific crystals. Since the substances tested are also present in other body fluids and in vegetable juices, the specificity of these tests has been questioned (lh). 

H18. Huggins, C., and Talalay, P., Sodium phenolphthalein phosphate as a substrate for phosphatase tests. J. Biol. Chem. 159, 399-410 (1945). 

El39 Ektachem Alkaline Phosphatase Test Methodology, MP2-37 (1984). 

Specimen tubes should be centrifuged with stoppers in place. Closure reduces evaporation, which occurs rapidly in a warm centrifuge with the air currents set up by centrifugation. Stoppers also prevent aerosolization of infectious particles. Specimen tubes containing volatiles, such as ethanol, must be stoppered while they are spun. Centrifuging specimens with the stopper in place maintains anaerobic conditions, which are important in the measurement of carbon dioxide and ionized calcium. Removal of the stopper before centrifugation allows loss of carbon dioxide and an increase in blood pH. Control of pH is especially important for the enzymatic measurement of acid phosphatase, which is labile under alkaline conditions engendered by CO2 loss. Indeed, once the serum is separated for acid phosphatase tests, a tablet of disodium citrate should be added to stabilize the pH at about 6.2. 

Several workers (28-29) reported an increase in the methylene blue reduction time when 0.05-0.5 lU Penicillin were present per milliliter of milk. Similarly, Manokidis et al. (30) noted that Penicillin and Oxytetracycline were responsible for a false positive phosphatase test in pasteurized or partially pasteurized milk while Streptomycin, Erythromycin and Neomycin inhibited the phosphatase test to some extent in partially pasteurized milk, but not in raw milk. Although raw milk containing antibiotic residues was never mistakenly identified as pasteurized, these authors suggested that as a "precaution" antibiotic assays be run in conjunction with the phosphatase test. 

Alkaline phosphatase Phosphatase enzymes are able to split specific phosporic acid esters into phosphoric acid and the related alcohols. Unlike most milk enzymes, it has a pH and temperature optima differing from physiological values pH of 9.8. The enzyme is destroyed by minimum pasteurization temperatures and therefore, a phosphatase test can be done to ensure proper pasteurization. 

NFPA Health 2, Flammability 1, Reactivity 2 Storage Store in freezer keep container tightly closed under inert atmosphere protect from light Uses Used to make blue indigo used in determination of prostatic acid phosphatase alkaline phosphatase test Manuf./Distrib. Fluka http //www.sigma-aldrich.com, Sigma... 

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

The dosage of flucytosine is 150—200 mg/kg orally in four portions every six hours. A 1% flucytosine solution has been developed for intravenous adrninistration. In some countries, a 10% ointment is also available. In patients with normal renal function, flucytosine is seldom toxic, but occasionally severe toxicity may be observed (leukopenia and thrombocytopenia). Plasma levels should be determined and the dose in patients with impaired renal function should be checked. Liver function tests (transaininases and alkaline phosphatase) should be performed regularly. In some patients with high flucytosine plasma levels, hepatic disorders have been observed (24). 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Table 32-3 summarizes laboratory results obtained on patients with three different causes of jaundice—hemolytic anemia (a prehepatic cause), hepatitis (a hepatic cause), and obstruction of the common bile duct (a posthepatic cause). Laboratory tests on blood (evaluation of the possibihty of a hemolytic anemia and measurement of prothrombin time) and on semm (eg, electrophoresis of proteins activities of the enzymes ALT, AST, and alkahne phosphatase) are also important in helping to distinguish between prehepatic, hepatic, and posthepatic causes of jaundice. 

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. 

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

Red blood cells also contain sufficient acid phenylphospha-tase for mild hemolysis to cause false elevations. Therefore, inhibitors such as ethanol, formaldehyde, copper sulfate> and 1-tartrate have been used to inhibit selectively the enzyme of one or more tissues and enhance the specificity of the test (101). Ethanol is unsuitable because it inhibits the enzyme from erythrocytes and prostate simultaneously, and because it yields serum activities which correlate poorly with prostatic disease. Formaldehyde inhibits the erythrocytic enzyme and has been said to yield clinically satisfactory results. The copoper resistant acid phosphatase of serum is elevated by metastatic carcinoma of the breast, as well as by other metastatic cancers, and is also elevated by a wide variety of non-cancerous diseases. 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

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