Protein Measurement with the Folin Phenol Reagent

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.

Use of p-nitrophenyl phosphate for assay of soil phosphatase activity

Publisher Summary This chapter discusses selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods for determining ascorbic acid are numerous. In general, chemical analyses for the vitamin are divided into two groups; the determination of the reduced form and the determination of the oxidized form. The former group of analyses is usually based upon the oxidation–reduction properties of ascorbic acid. These are widely used as the fundamental reactions in the measurement of vitamin C. The latter group of analyses is usually based upon the oxidation of the ascorbic acid and the subsequent formation of a hydrazone or a fluorophore. Best results are obtained if samples, especially plasma, are quickly stabilized with either trichloroacetic acid or metaphosphoric acid and immediately analyzed. Prompt stabilization is especially important in the case of plasma or serum. The greater stability of ascorbic acid in acid solution is because of the decreased tendency for the hydrolysis of the lactone ring with decreasing pH.

Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids

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.

https://jcp.bmj.com/content/jclinpath/7/4/322.full.pdf

Mary Jane Brock

Papers

A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum

The determination of vitamin A and carotene in small quantities of blood serum.

The quantitative determination of ascorbic acid in small amounts of white blood cells and platelets.

The interrelationship of dietary, serum, white blood cell, and total body ascorbic acid.