Evaluating the activity and stability of sonochemically produced hemoglobin-copper hybrid nanoflowers against some metallic ions, organic solvents, and inhibitors

The disadvantage of the conventional protein-inorganic hybrid nanoflower production method is the long incubation period of the synthesis method. This period is not suitable for practical industrial use. Herein, protein-inorganic hybrid nanoflowers were synthesized using hemoglobin and copper ion by fast sonication method for 10 min. The synthesized nanoflowers were characterized via scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, and Fouirer-transform infrared spectroscopy. The activity and stability of the nanoflowers in the presence of different metal ions, organic solvents, inhibitors, and storage conditions were also evaluated by comparing with free hemoglobin. According to obtained results, the optimum pH and temperatures of both hybrid nanoflower and free hemoglobin were pH 5 and 40 °C, respectively. At all pH levels, nanoflower was more stable than free protein and it was also more stable than the free hemoglobin at temperatures ranging between 50 °C and 80 °C. The free protein lost more than half of its activity in the presence of acetone, benzene, and N,N-dimethylformamide, while the hybrid nanoflower retained more than 70% of its activity for 2 h at 40 °C. The hybrid nanoflower activity was essentially increased in the presence of Ca²⁺, Zn²⁺, Fe²⁺, Cu²⁺ and Ni²⁺ (132%, 161%, 175%, 185% and 106%, respectively) at 5 mM concentration. The nanoflower retained more than 85% of its initial activity in the presence of all inhibitors. In addition, it retained all its activity for 3 days under different storage conditions, unlike free hemoglobin. The results demonstrated that new hybrid nanoflowers may be promising in different biotechnological applications such as catalytic biosensors and environmental or industrial catalytic processes.