A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum
01 Jul 1946-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 164, Iss: 1, pp 321-329
TL;DR: A rapid method has been devised which requires only 5 c.mm.
About: This article is published in Journal of Biological Chemistry.The article was published on 1946-07-01 and is currently open access. It has received 2921 citations till now. The article focuses on the topics: Alkaline phosphatase & Blood serum.
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TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: In this paper, a simple method of assaying soil phosphatase activity is described, which involves colorimetric estimation of the p-nitrophenol released by the enzyme when the soil is incubated with buffered (pH 6·5) sodium pnphosphorus solution and toluene at 37°C for 1 hour.
Abstract: A simple method of assaying soil phosphatase activity is described. It involves colorimetric estimation of the p-nitrophenol released by phosphatase activity when soil is incubated with buffered (pH 6·5) sodium p-nitrophenyl phosphate solution and toluene at 37° C for 1 hr. The method is rapid and precise, and it has significant advantages over methods previously proposed for assay of soil phosphatase activity.
3,503 citations
TL;DR: The conditions under which enzymic hydrolysis is carried out are the same as in the King-Armstrong method, as modified by King (1951), the amino-antipyrine reagent (A.A.P.) being used to estimate the phenol liberated.
Abstract: Plasma phosphatase was first determined by Martland (1925) and then by Kay (1930), using glycerophosphate and estimating the inorganic phosphate liberated at 370 C. at a specified pH and in a given time. The procedure has been variously modified and simplified by Jenner and Kay (1932), Bodansky (1933), Shinowara, Jones, and Reinhart (1942). Several substrates, other than glycerophosphate, have also been used, e.g., phenyl phosphate (with determination of either the liberated phenol, King and Armstrong, 1934, or phosphate, King, Abul-Fadl, and Walker, 1951), nitro-phenyl phosphate (by the colour of the liberated nitrophenol, Ohmori, 1937; King and Delory, 1939; Bessey, Lowry, and Brock, 1946), naphthyl phosphate (by determination of naphthol by a diazo method, Seligman, Chauncey, Nachlas, Manheimer, and Ravin, 1951), phenolphthalein phosphate (by the colour of alkaline phenolphthalein, Bray and King, 1943; Huggins and Talalay, 1945). Of the.se procedures, the Bodansky (glycerophosphate) and the King-Armstrong (phenyl phosphate) are perhaps the most widely used. King and Armstrong (1934) determined free phenol, liberated from phenyl phosphate by the phosphatase, by either the Folin and Ciocalteu (1927) phosphomolybdic reagent or by the diazo reaction, and preferred the former. Both procedures required the precipitation and removal of the plasma proteins subsequent to the hydrolysis of the substrate. In 1951 Grifols Lucas proposed a procedure for determination of the free phenol, which did not require removal of the proteins. This was based on the 4-amino-antipyrine reaction of Gottlieb and Marsh (1946). In this method the conditions under which enzymic hydrolysis is carried out are the same as in the King-Armstrong method, as modified by King (1951), the amino-antipyrine reagent (A.A.P.) being used to estimate the phenol liberated. 4-Amino-antipyrine reacts with certain phenolic substances in the presence of alkaline oxidizing agents to produce quinonoid substitution products. These give a red colour proportional to the phenol present, and can be determined colorimetrically. Grifols-Lucas found that A.A.P. does not react with plasma proteins, and their removal is unnecessary when phosphatases are estimated.
1,665 citations
TL;DR: Micromethods have been developed and 35S-labeled chondroitin sulfates A, B, and C in a given mixture have been precisely and rapidly determined by measuring radioactivity alone.
1,314 citations
TL;DR: In this paper, an improved method to assay activities of α- and β-glucosidases and α-and β-galactosidase in soils is described.
Abstract: An improved method to assay activities of α- and β-glucosidases and α- and β-galactosidases in soils is described. It involves extraction and colorimetric determination of the p-nitrophenol released when 1 g of soil is incubated with 5 ml of buffered p-nitrophenyl glycoside solution at 37°C for 1 h. The reagents [0.5 M CaCl2 and 0.1 M Tris (hydroxymethyl)aminomethan THAM, pH 12] used for extraction of the p-nitrophenol released give quantitative recovery of p-nitrophenol added to soils and do not cause chemical hydrolysis of the substrates. Results showed that these enzymes have their optimum activities at buffer pH 6.0. The initial rates of p-nitrophenol release obeyed zero-order kinetics. β-Glucosidase activity was the most predominant of the four enzymes. The temperature dependence of the rate constant conformed to the Arrhenius equation up to the point of enzyme inactivation (60°C for α- and β-galactosidases and α-glucosidase and 70°C for β-glucosidase). The average activation energy values of these enzymes in three soils were 43.1, 30.8, 57.0 and 32.6 kJmol−1 for α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase activities, respectively. By using the Lineweaver-Burk plot. the Km values were the lowest for β-glucosidase activity. The Vmax values varied among the four enzymes and soils studied.
1,156 citations
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