SENSITIVE DETERMINATION OF NUCLEOTIDES AND POLYNUCLEOTIDES BASED ON THE FLUORESCENCE QUENCHING OF THE TB3-TIRON COMPLEX()

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 nucleoti des and polynucleotides to inhibit the formation of a strongly fluores cent complex of Tb3+ ion with Tiron. The possibilities of spectrofluor imetric measurements of these systems were studied under optimal condi tions (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 u sed as a fluorescence test for the phosphate moieties of nucleotides a nd 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 replica tes) are within 4.0% in the middle of the linear range. The fluorescen ce quenching mechanism of these systems is also discussed.