Fluorometric investigation of boric acid-pyridoxal 5′ phosphate interaction: A turn-on-fluorescence assay for the detection of boric acid in water bodies and fetal bovine serum

Abstract

Boron is a trace element that is beneficial to plants as well as human and animal health. Boron supplementation is beneficial for absorbing vitamin D and magnesium, activating antioxidant enzymes and anticancer treatments. However, environmental accumulation of boron exceeding the permissible limit harms plants, animals and humans. Boric acid (BA), a form of boron, is known to interact with biomolecules such as NAD+/NADH, vitamin C, vitamin D, vitamin B1 and vitamin B6 determined through techniques like NMR, mass spectrometry, and FTIR. The fluorescence interaction of BA with the biologically active form of vitamin B6, Pyridoxal 5′ phosphate (PLP), has not been explored to date. Therefore, this study reports the interaction mechanism of BA with PLP via fluorescence spectroscopy analysis. The absorption, excitation and fluorescence lifetime measurements indicate that BA may form a complex through the mono or di-esterification of the phenolic hydroxyl group and aldehyde of PLP at different pH, forming the boronate esters. BA addition enhanced the fluorescence intensity of PLP by 12–22-fold in phosphate buffer (at acidic, neutral, and basic pH conditions). On the other hand, in citrate buffer at pH 4.0, BA enhanced PLP fluorescence by 28-fold. The binding constant for BA in phosphate buffer and citrate buffer was found to be 173 mol−1·L and 623 mol−1·L, respectively. At the same time, the detection limit for BA was found to be 445 μg/L and 212 μg/L in respective buffers. Therefore, PLP was used as a turn-on fluorescent probe to detect and quantify BA in water bodies and fetal bovine serum (FBS). The developed method successfully detected BA in environmental water bodies, achieving an average recovery rate of 99.84% (with a relative standard deviation (RSD) of 0.38%) in phosphate buffer and 81.59% (RSD, 4.56%) in citrate buffer. Furthermore, detecting BA in PLP-spiked FBS has been demonstrated in citrate buffer at pH 4.0, yielding an average recovery rate of 108.25% (RSD, 3.19%). Therefore, this study provides a basis for understanding the interaction of biomolecules like vitamins with boronates which aid in development of novel boronate based therapeutics and also helps in understanding the metabolic reactions of vitamin derivatives using fluorescence spectroscopy.