Conjugation of nattokinase and lumbrukinase with magnetic nanoparticles for the assay of their thrombolytic activities

Two important thrombolytic enzymes, nattokinase (NK) and lumbrukinase (LK), were immobilized onto fine magnetic Fe3O4 nanoparticles using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) as the coupling reagent, and their thrombolytic activities were studied. The Fe3O4 nanoparticles and NK- and LK-conjugated magnetic nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, X-ray diffraction, and UV–vis absorption spectroscopy. Dual kinetic absorbance measurements at 405 and 630 nm were employed to measure their thrombolytic activity. Analysis of protein amount showed that the optimum conditions for NK and LK binding to nanoparticles were respectively at a mass ratio of 2:1:1, 2:1:2 (magnetic nanoparticles:protein:EDC), and pH 6.00. Thrombolytic activity assay showed that the best thrombolytic activity could reach 91.89% for NK–nanoparticle conjugates and 207.74% for LK–nanoparticle conjugates, which are much higher than the pure enzymes (NK, 82.86%; LK, 106.57%).