Orcinol assay

The only method used to quantitate ribonucleic acid is the orcinol assay.20 With this method, RNA is depurinated in concentrated HC1 and the resulting ribosephos-phates are dephosphorylated and dehydrated to produce furfural (Eq. 1.5). Furfural then reacts with orcinol in the presence of Fe3+ to yield colored condensation products, as shown in Eq. 1.4, which together possess an absorption maximum at 660 nm. 

The original orcinol assay has been modified to improve its selectivity toward RNA over DNA and sugars. The improved method21 uses a 1.0-mL RNA sample incubated 24 h at 40 °C with 4.0 mL of 85% sulfuric acid prior to addition of the orcinol reagent (containing no Fe3+). Under these conditions, colorimetric measurements are made at 500 nm, where products formed by the reaction of levulinic acid (from DNA) with orcinol do not absorb. Proteins (e.g., BSA) do not interfere, and the method is sixfold more selective to RNA over DNA than the original orcinol assay. 

The method has advantages over the resorcinol and orcinol assays by virtue of its sensitivity and flexibility in determining free and bound sialic acid, and also in the lower level of interference from other monosaccharides and other substances (JOURDIAN tz/. 1971). 

It is important to be able to determine which chromatography column fractions contain polysaccharides and, specifically, which fractions contain hexosyl, pentosyl, or uronosyl residues. It is also important to detect in the fractions the presence of proteins and the presence of the specific amino acid characteristic of wall proteins, hydroxyproline. The detection of these substances is carried out by facile and sensitive colorimetric procedures. Although these reactions are not discussed here, the most frequently used colorimetric assays in our laboratory are the anthrone assay for detection of hexosyl residues 50), the orcinol assay for detecting pentosyl residues 50), the m-hydroxy-diphenyl assay for detection of uronosyl residues 35), the Lowry assay for detection of proteins 90), and the Kivirikko and Liesmaa assay for the detection of hydroxyprolyl residues 76). 

Thin-layer chromatography has been used by Foppiano and Brown229 to assay the total neomycin B and C content of neomycin sulphate. The neomycin zone was scraped off the plate and reacted with orcinol/ferric chloride reagent, the absorbance of the resulting colour being measured at 665nm. By this procedure a precision of - 2% was achieved. 

Minimum amounts of 2-3 /ig of sialic acids can be determined in, for example, the orcinol-Fe3+ assay, if the volumes of the Bial reagents23 are one-fifth of those originally described.107 By using this method, sialic acids can be accurately determined only if they have been prepurified, as free or glycosidically bound pentoses, hexoses, or alduronic acids interfere with the reaction by giving green chromo-phores.107 In our experience, an approximate determination of the sialic acid in a complex carbohydrate is only possible if its sialic acid content is >5%. 

The X-ray crystal structure of A1 -tetrahydrocannabinolic acid B is reported,362 and one of the previously reported (Vol. 4, p. 75) dihydrobenzofurans from the citric acid-catalysed condensation of orcinol with menth-4-en-3-ol is shown to be (256) by X-ray analysis.363 G.c.-m.s. assay of A -THC-OMe allows the detection of 1 ng mP1 plasma of A -THC.364 The mass spectral fragmentation of A -THC, A6-THC, and some isomeric cannabinoids to the prominent m/e 231 ion has been examined.365 Miniaturized syntheses of 32 natural, or potentially natural, cannabinoids are reported in connection with their chromatographic analysis.366... 

Figure 2-17. Reactions involved in the assay of nucleic acids by the orcinol procedure.

Figure 2-18. Absorbances observed with increasing amounts of RNA assayed by the orcinol reaction.

Although several colorimetric methods have been developed in the past, only two main procedures are currently routinely applied, namely the orcinol/Fe /HCl assay, known as the Bial reaction, and the periodie acid/thiobarbituric acid assay. For microadaptations of these two different tests, see ref. [11]. 

In the first assay, the sample is mixed with orcinol, FeCls and concentrated HCl and heated at 96°C. The formed purple to red-violet chromophore is extracted with isoamyl alcohol and its absorbance measured at 572 nm. Because of the use of HCl, the method can be used to quantitate the total amount of both free and glycosidically bound sialic acids. Due to the strongly acidic conditions, ester groups are released. As the assay does not discriminate between bound and free sialic acids, it is widely used to monitor the presence of sialic acids in either form during fractionation of biological material. It should be noted that other monosaccharides, especially pentoses, hexoses and uronic acids interfere with the assay, which is of importance when small amounts of sialic acid are present. 

All fractions are made up to measured volumes and aliquots are removed for analysis. RNA may be measured by the orcinol reaction and DNA by the di-phenylamine reaction, the values obtained being referred to those obtained upon standard solutions of pure RNA and DNA, respectively. (In the S method, these are determined in the one and only nucleic acid fraction.) In addition, total and inorganic phosphorus in each fraction may be measured. The assay procedures are described in detail in Section III. 

Step 4. Treatment with hot acid releases all the adenine and guanine (99) and some desoxyribose and inorganic phosphate from DNA, the remainder appearing as acid-soluble pyrimidine-nucleotide combinations (36). Diphenylamine reacts only with the free desoxyribose or purine de-soxyribosides. Orcinol reacts slightly with DNA hence its use for RNA assay in the presence of large amounts of DNA requires a correction (93). 

Detailed colorimetric assay methods for nucleic acid derivatives will be confined to descriptions of slight modifications of three procedures diich have withstood well the test of time (f) the Mejbaum (76) modification of the Bial orcinol reaction for pentose (2) the Dische (42) diphenylamine method for desoxypentose and (S) the method of Fiske and Subbarow (45) for inorganic orthophosphate. Many other modifications of the original techniques have been advanced but, in our opinion, none offer a complete solution of such difficulties as may be encountered in nudmc acid studies. Discussions of the colorimetric techniques will be foimd in Schlenk s review (89), in the manual edited by Umbreit cf oZ. (61), and in the review by Dische (41). 

Reports of sialic acids in plants exist in the literature (Mayer et al. 1964, Onodera et al. 1966), but the analytical methods employed were insufficient to exclude other compounds such as 2-keto-3-deoxy acids noted earlier in this chapter. Several investigations produced negative results, although a reaction in the periodic acid/thiobarbituric acid assay was obtained. The compounds in question gave no colour in the direct Ehrlich, orcinol/Fe and resorcinol assays for sialic acids (Gielen 1968, Cabezas 1968, 1973, CABEZAsand Feo 1969, Unger 1981). 

Those colorimetric methods developed at this time and found to be suitably sensitive and specific are the orcinol and resorcinol methods (see II. 1 below) and the periodic acid/thiobarbituric acid assay (see II. 2). These methods together with a recently introduced assay using methyl-3-benzothiazolinone-2-hydrazone and fluorimetric methods (II. 3 and II. 4, respectively) form the basis of currently used techniques. 

As with the orcinol/Fe assay, both free and bound sialic acids are measured by this method and no discrimination can be made between these forms. 

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