Spectrofluorometric Determination of Trace Amounts of Coenzyme II Using Norfloxacin-Terbium Complex as a Fluorescent Probe

Abstract

The pyridine nucleotides (NAD and NADP) are natural coenzymes present in all animal and plant organisms. They are essential to the function of many catabolic and biosynthetic pathways found within living organisms. Each of these forms has a particular role in enzyme regulation during differentiation and similarity processes.The familiar methods for the determination of NADP include electrochemical methods, 1-8 HPLC, 9-17 microfluorometric method, 18 bioluminescence method, 19 spectrophotometric method, 20 enzyme-linked immunosorbent assay, 21 capillary electrophoresis method 22 and FIA-CME. 23,24 But there was no report about a spectrofluorometric method for determination of NADP using Tb 3+ -NFLX as fluorescent probe.Norfloxacin is one kind of bacteriophages that contains α-carbonyl carboxylic acid configuration. Some researchers used the highly sensitive fluorescence of Tb 3+ and norfloxacin or ciprofloxacin complexes to determine these bacteriophages [25][26][27][28] showing high sensitivities. In this work, we choose norfloxacin as the ligand of Tb 3+ and investigated the possibility of the enhancement of the Tb 3+ fluorescence sensitized by it and using NADP as co-ligand. Thus, we established a method and successfully applied it to determine NADP synthetic water samples with satisfactory results. It is simple and relatively free of interference from coexisting substances. The mechanism of fluorescence enhancement between Tb 3+ -NFLX complex and NADP was also studied. Experimental ReagentsAll chemicals used were of analytical-reagent or higher grade. Doubly distilled demineralized water was used for the preparation of all solutions and for all determinations. ; pH, 7.60. ApparatusWe used a pHs-3C digital pH meter (Shanghai Leici Device Works, China), an RF-540 spectrofluorometer, and a UV-265 spectrophotometer (Shimadzu, Kyoto, Japan). General procedureTo 10 ml color comparison tubes, solutions were added in the following order: 2.0 ml 1.53 × 10 -5 mol l -1 NFLX solution, 2.0 ml 1.11 × 10 -5 mol l -1 NADP solution, 1.5 ml 5.0 × 10 -5 mol l -1 Tb 3+ solution and 1.5 ml buffer solution. Each mixture was diluted to the mark with water and kept for 15 min at room temperature. The fluorescence intensity was measured at λex/λem = 335 nm/545 nm. The enhanced fluorescence intensity of Tb 3+ -NFLX by NADP was represented as ∆F = F -F0. Here F and F0 are the fluorescence intensities of the systems with and without NADP, respectively. Results and Discussion Characteristics of excitation spectraThe fluorescence excitation spectra are shown in Fig. 1. From curve 3 it can be seen that single Tb 3+ solution has nearly no peak. Comparing curve 3 with curve 6, one sees that, after the addition of NFLX into the Tb 3+ solution, NFLX can form a binary complex with Tb 3+ that leads to energy transfer from Tb 3+ to NFLX. Two little characteristic peaks of Tb 3+ appear at 490 nm and 545 nm, corresponding to the 3 D4-7 D6 and 3 D4-7 D5 transition of Tb 3+ , respectively. Comparing curve 7 with curve 6, one can see that the characte...

Luminescent Chemical and Physical Sensors Based on Lanthanide Complexes

“…The same is observed in case of NADP [59], DNA [60], heparin [61], GTP, and GDP [62]. On the other hand, the emission is quenched by phosphate [59], pyrophosphate [63], and lecithin [64].…”

“…The same is observed in case of NADP [59], DNA [60], heparin [61], GTP, and GDP [62]. On the other hand, the emission is quenched by phosphate [59], pyrophosphate [63], and lecithin [64]. These responses can be exploited to monitor enzyme activities and was applied to screening assays for enzyme inhibitors, e.g., for adenylyl cyclases [63], and for the determination of GTPase activity of Ras proteins [62].…”