Orcinol reaction

Amperometric parameters have been studied and optimized for sugar separations.57236 The orcinol reaction has also been used as a post-column detection system for sugars.237... 

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

A sample from each fraction is transferred to a cuvette and its absorbance at 260 nm is determined. Data from this type of experiment appear in Figure 4-17. The orcinol reaction may be used to identify the nucleotide elution peaks (see Chapter 2 for the appropriate procedures). 

The column is washed with borate-saline until all the 2-ME is removed, with 0.2 M galactose in borate-saline buffer until dgRTB/dgRT impurities are removed, and with borate-saline buffer until all galactose is removed (determined using the orcinol reaction). 

The mechanically fragmented brain or spinal cord was dehydrated with acetone, as in the isolation of cerebrosides.7 Gangliosides are present in the water-soluble fraction they were recognized by color reactions (for example, Bial s orcinol reaction) based on the presence of sialic acid.159 The ganglioside mixture may be purified by partition... 

The orcinol reaction (Al) is simple, and further evidence of identification can be gained by determining the absorption spectrum of the color obtained by the reaction. The reaction is not specific for pentoses. Bial s test (HI) for pentoses in the urine represents the qualitative version of this reaction. 

Co- ph is a basic -watersoluble substance, resistant to heat in nUralin solution and to oxidation with HjOi or alkaline permanganate, but fairly unstable in acid solutions HCl inactivates in 5-10 min. at 100° C. and in 1 hr. at room temperature). Activity is markedly impaired by treatment with NaOI, NaOBr, KiFe(CN)6 or Na2SOt. The concentrates are still heavily contaminated with creatine and pve a positive orcinol reaction for pentose. (Do-aph could not be replaced by ashed concentrate or by any known enzyme activator or muscle extractive. Further investigap tion of as-aph and its coenzyme had been suspended in view of war-time conditions. 

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. 

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). 

Nucleic acid synthesis. A striking observation first made by Cohen (52,54) is that in cells infected with T2r+ only DNA, the nucleic acid found in the virus, is synthesized in contrast to normal bacteria which make about 3 times as much RNA as DNA. RNA when measured as pentose by the orcinol reaction remains constant. This phenomenon is clearly of paramount importance. It has been confirmed for T6 bacteriophage by Kozloff (private communication). However, Herriott s data (129) on E. coli infected with T2 give indication of a fall in RNA. Although protein synthesis is apparent from the moment of infection, DNA synthesis begins only after a lag of 7 to 10 minutes (54). This holds for T4 and T6, as well as T2 (62). As shown in Fig. 6, DNA increases linearly with time after a lag of about 10 minutes and finally levels off at about 100 minutes. Doermann and Cohen (unpublished experiments... 

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