Reagent-Free Spectroscopic Assays for Arginase Activity for Heterogenic System

Arginase is an enzyme of medical significance that plays a crucial role in research on macrophages, in the field of oncology, and serves as a biomarker in various areas. However, the current methods for assessing its activity rely on indirect, time-consuming colorimetric assays, which cannot provide real-time kinetic data in complex media. This is a critical aspect in biomedical research. To address this issue, we have developed two novel spectroscopic methods without the use of reagents, based on circular dichroism (CD) spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The approach based on CD proved to be particularly effective in capturing the complete progress curve of arginine hydrolysis within a single measurement, with high sensitivity, enabling detection limits as low as 0.026 U/mL. Nonetheless, the use of CD is primarily restricted to homogeneous and optically transparent solutions. In contrast, ATR-FTIR allows for real-time monitoring of specific bands within the substrate and product (arginine and ornithine, respectively) in complex and non-transparent media such as cell cultures, without the need for sample preparation. This method enables us to measure arginase activity in a living culture of Lewis lung carcinoma cells (LLC1), where the liposomal form of arginase demonstrates a remarkable 6.4-fold increase in specific activity compared to the native enzyme.