Magnetic nanoparticles coated with immobilized alkaline phosphatase for enzymolysis and enzyme inhibition assays

Abstract

Magnetic nanoparticles are potentially useful as supports for biomacromolecules because of their biocompatibility, low toxicity and easy separation. In this study, alkaline phosphatase (ALP) was used as a model enzyme, and a new type of immobilized ALP was prepared on superparamagnetic nanoparticles and confirmed by various characterization techniques. X-ray diffraction (XRD), scanning electron microscopy (SEM) and vibrating sample magnetometry (VSM) results present that the synthesized nanoparticles possess a clear three-dimensional core-shell architecture with an average diameter of about 390 nm and a high saturation magnetization of 86.7 emu g^-1. Fourier-transform infrared spectra (FTIR) and thermogravimetric analysis (TGA) results show that ALP was successfully attached to the surface of magnetic nanoparticles via a crosslinking technique. An enzyme inhibition study was performed on the immobilized ALP magnetic nanoparticles using theophylline, l-tryptophan and d-tryptophan as model inhibitors. The enzyme kinetics indicate that dl-tryptophan possesses chiral discrimination inhibition and l-tryptophan exhibits uncompetitive inhibition for ALP, compared with no obvious inhibition observed with its enantiomer. The results also show that theophylline is a noncompetitive inhibitor and has a markedly higher inhibitory effect than l-tryptophan. The protocol described allows easy manipulation, reduces procedural time and can be adapted to high-throughput screening of enzyme reactions and inhibitors.