Disodium p-nitrophenyl phosphate

Randolph et al. (1985) Batch Hydrolysis of disodium p-nitrophenyl phosphate to p-nitrophenol Alkaline phosphatase... 

In 1947, Hudson et al. (H15) developed a method for acid phosphatase which, like the procedure of Bessey et al. for alkaline phosphatase (B16), was based upon the use of p-nitrophenyl phosphate as substrate. The buffer substrate solution consisted of equal volumes of a 0.1 M sodium acetate-acetic acid buffer, pH 5.4, and 0.001 M magnesium chloride and of a 0.4% solution of approximately 50% pure disodium p-nitrophenyl phosphate in 0.001 N HCl. To 1 ml of this solution, 0.1 ml of the serum sample was added. The final concentrations in this reaction mixture were 0.045 M acetate buffer, pH 5.4 magnesium chloride. 0.00045 ilf substrate, 0.004 M. The reaction was allowed to run for 30 minutes at 38°C, and the reaction was stopped by the addition of sodium hydroxide. The liberated yellow p-nitrophenol was read at 400 nm and the amount was... 

The phenotypes of erythrocyte acid phosphatase not only exhibit differences in electrophoretic behavior, but also show variation in total acid phosphatase activity. Spencer et al. (S26) studied the distribution of red cell phosphatase activities in hemolysates from 275 individuals with various phenotypes. The assay was performed with 0.01 M disodium p-nitrophenyl phosphate as substrate at pH 6.0 in citrate buffer. The units of activity were expressed as /unoles of p-nitrophenol liberated in 0.5 hour at 37°C per gram of hemoglobin. These results are shown in Table 5. 

Substrate tablets 5 mg tablets of disodium p-nitrophenyl phosphate (PNPP) (e.g., Amresco, Solon, OH, or Pierce Chemical, Rockford, IL) (see Note 10). 

Randolph s tests with alkaline phosphatase were carried out in a stirred autoclave. An amount of the disodium salt and some water, which is required for the enzyme catalyzed hydrolysis, were placed in the autoclave along with a sealed glass ampule containing the enzyme. In this case water is necessary not just to render the enzyme active, as Klibanov found, but also to serve as a reactant in the hydrolysis. Carbon dioxide was admitted, the temperature and pressure adjusted to the level desired, and the sealed ampule shattered to expose the enzyme and to mark the zero point of the reaction sequence. In their studies they investigated the effects of changing the relative amount of enzyme on the rate of conversion of the disodium salt of p-nitrophenyl phosphoric acid to p-nitrophenol. They measured the amount of conversion by UV analysis of the solution removed from the autoclave at the end of a reaction test. The results are shown in Figure 11.1 based upon these results and other experimental results, the authors concluded that the rate-determining step of the enzyme-catalyzed reaction was the dissolution of disodium p-nitrophenyl phosphate in supercritical carbon dioxide. 

Alkaline phosphatase Polymerizations CO2 disodium p-nitrophenyl phosphate 100 bar 35 °C [1]... 

The earliest reported demonstration of enzymatic activity in a supercritical fluid was for the reaction of disodium p-nitrophenyl phosphate to p-nitro-phenol, catalysed by alkaline phosphatase. Randolph et aL [26] detected the product in yields of up to 71% in carbon dioxide at 35°C and 100 atm, in the presence of 0.1% v/v water. Hammond et al. [33] found tyrosinase, a polyphenol oxidase, to be catalytically active for the oxidation of 4-methyl phenol in both supercritical carbon dioxide at (36 2)°C and supercritical trifluoro-methane at (34 2)°C, with oxygen, at a total pressure of 345 bar. Use of a flow reactor permitted isolation of greater quantities of the catecholic product (1,2-dihydroxy, 4-methylbenzene). Oxidative activity for 4-chlorophenol substrate was appreciably lower. 

The enzyme alkaline phosphatase is naturally present in milk. As it is sensitive to heat it is used to measure the effectiveness of pasteurization. Milk is diluted with a buffer (pH 10.6) containing disodium phenol phosphate which is hydrolyzed by the enzyme releasing phenol. The phenol is reacted with 2,6-di-bromoquinonechloroimide producing a color that is measured spectrophotometrically at 610 nm. An earlier version of this test uses p-nitrophenyl phosphate and the amount of yellow p-nitrophenyl which is released is compared with standard comparator disks. 

Add 10 0 pL of p-nitrophenyl phosphate lOOx stock substrate solution (lOOx stock is made by adding 40 mg of 4-nitrophenyl phosphate disodium salt hexahydrate into 10 mL of ddHjO). 

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