A Minimal Hemin-Albumin Nanozyme with Peroxidase-like Activity Enabling Tyrosine-Crosslinked Hydrogel Formation

Heme enzymes catalyze key oxidative reactions, yet their use in biotechnology is often limited by high production costs, low stability, and intricate operational conditions. Hemin, the catalytic iron-porphyrin cofactor of heme enzymes, has been explored as an alternative. However, its practical use is limited by its aggregation and deactivation in aqueous media. Here, we report a minimal biomimetic nanozyme based on the stabilization of hemin with bovine serum albumin (BSA) to enable aqueous catalysis and hydrogel formation under mild aqueous conditions. Through alkaline-mediated synthesis, hemin was stabilized with BSA, allowing the generation of a Hemin@BSA noncovalent hybrid with peroxidase- and catalase-like activity, outperforming free hemin or BSA complexed with hemin prepared under physiological conditions. Beyond standard peroxidase assays, Hemin@BSA catalyzed the oxidative C-C coupling of N-acetyl-tyrosine (NAT), yielding dityrosine as the main product. This enzymatic-like property was successfully applied at the macromolecular level, enabling the crosslinking of hyaluronic acid-tyramine (HA-TyrA) conjugates, yielding the formation of hydrogel networks with mechanical properties comparable to HRP-mediated crosslinked hydrogels. The cytotoxicity of hemin and Hemin@BSA was evaluated in both U87 glioblastoma and normal human astrocytes (NHA) cells, while cellular uptake and reactive oxygen species (ROS) generation were investigated in U87 cells, demonstrating efficient cellular uptake of Hemin@BSA and higher ROS levels induced by free hemin compared with Hemin@BSA. Overall, simple heme-protein complexes lie as the interface between molecular catalysts and functional biomaterials. They provide a low-cost and robust alternative to natural peroxidases, making them promising tools for creating redox-based hydrogels towards biomedical applications.