Sensitive determination of nucleotides and polynucleotides based on the fluorescence quenching of the Tb3+-Tiron complex
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A sensitive method using fluorescence quenching for the determination of nucleotides (ATP, ADP, AMP, CTP, UTP) and polynucleotides [poly(A), poly(I), poly(U)] is proposed. It is based on the ability of nucleotides and polynucleotides to inhibit the formation of a strongly fluorescent complex of Tb3+ ion with Tiron. The possibilities of spectrofluorimetric measurements of these systems were studied under optimal conditions (pH 6.9 in hexamethylene tetramine-HCl buffer, 1.2 x 10(-6) mol/L of Tb3+, 4.0 x 10(-6) mol/L of Tiron, lambda(ex) = 317 nm, lambda(em) = 546 nm). The results showed that the Tb3+-Tiron complex could be used as a fluorescence test for the phosphate moieties of nucleotides and polynucleotides. The detection limits are 0.3, 1.2, 3.7, 0.2, 0.3, 1.1, 0.6 and 0.9 ng/mL for ATP, ADP, AMP, CTP, UTP, poly(A), poly(I), and poly(U), respectively. The relative standard deviations (6 replicates) are within 4.0% in the middle of the linear range. The fluorescence quenching mechanism of these systems is also discussed.