Sensitive,
Selective, and Simple Determination of Adenine
Content in Nucleic Acids by Fluorometric Method
Nucleic acids contain four main bases, but their ratio differs according to their origins. The content of each base has usually been determined by ultraviolet absorption method after hydrolysis (l-4,) of nucleic acids and separation of bases by chromatography
(5-7). This method is rather t,imeconsuming because it involves a procedure of chromatographic separation. L>evclopmcnt of a more sensitive and simple analytical method is worthwhile for biochemical investigation.
Previously, we reported a fluorometric determination of adenine and its derivatives by reaction with glyoxal dihydrate trimer in an acidic medium (8 I. This method is sensitive, and specific for ndenine, so it was expected that adenine content in nucleic aci(Is might simply have been determined without separation from other bases. In addition, the hydrolysis and fluorescence-l)rodrlciiig reaction could t)e performed in one process. This paper deals with t,he fluorometric analysis of adenine content in nucleic acids.
ildeninc was from Tnkarakosan Co., Lt,d. Glyoxal dihydrate trimer, adenosin&'-phosphate
(5'-AMP), and 2'-dcosyadenosine-5'-phosphate (5'-deAMP) wore from Sigma Chemical C'ompany. Acetic acid, hydrochloric acid, and I)errhlorir acid (reagent gradej were from Wako Pure Chemical Co., T,ttl. Origins of DS,4 anI1 RNA were specified in Figures and Table intlividunlly.
l~csaltctl distillctl water was used throughout this experiment.
Fluorescence intensity was measured in :I Hitachi fluorescence spectrophotomctcr MPF-2A.
Reagents. N Hydrochloric acid. Glacial acetic acid (purified by distillation 1. Glyoxal diliydrate trimer solution (21 g glyoxal diliydrate trimer was dissolved in 100 ml water; a slight fluorescence of the solution, if any, was removed by passing through a carbon column ) Procedure.