BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT ZEBRAFISH URATE OXIDASE: KINETIC AND THERMAL STABILITY ANALYSIS

The protein urate oxidase (UOX) is the first enzyme in the uric acid catabolism pathway. Its structure is a homotetramer, formed by the association of two dimers containing a disulfide bond between them. In humans, the UOX gene was inactivated by a series of nonsense mutations, resulting in increased serum urate concentrations and, in more severe cases, hyperuricemia. Enzyme-based therapies are commonly used to treat this disorder, including rasburicase and pegloticase. However, rasburicase is associated with high immunogenicity, which restricts its use in prolonged treatments, while pegloticase, a PEGylated biologic, remains costly to produce and is therefore inaccessible to a substantial portion of patients — underscoring the need for more affordable enzyme-based alternatives. Given these limitations, identifying alternative urate oxidases remains an important yet insufficiently explored area. In this regard, UOX derived from zebrafish (Danio rerio) emerges as a promising candidate, as this organism is widely used as a model system and is evolutionarily closer to vertebrates, including humans, than microbial sources, which may favor biological compatibility. Despite these favorable aspects, there is still a lack of information on some physicochemical properties of zebrafish UOX, such as its stability in solution. The aim of this study was to analyze and characterize the thermal stability of recombinant zebrafish UOX by comparing its kinetic behavior before and after different heat treatments. In this work, we analyzed by UV-Vis spectrophotometric assays, the kinetic profile of the enzyme after incubation at 22, 37, and 55 °C. In addition, we evaluated whether heat-induced protein aggregation affected enzymatic activity, comparing kinetic behavior in samples with and without visible precipitates. We are also investigating different additives that may extend the shelf life of this enzyme. Taken together, these results indicate that zebrafish UOX warrants further investigation as a potential alternative to currently available therapeutics for hyperuricemic diseases.