生体試料中の活性プロテアーゼの検出と定量：最適化されたプロトコル

Matrix metalloproteinases (MMPs) are enzymes that degrade extracellular matrix (ECM) proteins and activate cytokines and chemokines, playing a critical role in tissue remodeling. Monitoring MMP activity is important for diagnosing and tracking diseases, studying disease progression, and developing new diagnostic and therapeutic strategies. This article highlights methods for detecting active gelatinases, specifically MMPs-2, -7, -9, and -13 in various biological samples.

The described protocol utilizes an electrophoresis-based biochemical technique commonly used for protein analysis, with the key modification of incorporating a specific substrate, such as gelatin or casein, into the gel. This method, known as zymography, is named according to the substrate used. For example, it is called 'gelatin zymography' when gelatin is used as the substrate.

When performing zymography, it is crucial to account for the amount of proteinase in different samples, such as plasma which contains significantly higher concentrations of active MMPs compared to other body fluids, tissues, or cells. As a result, only small volumes of plasma are required to produce distinct bands in the zymography gel. Additionally, our findings show that MMP activity, especially active MMP-9, is significantly higher in sonicated samples compared to non-sonicated samples. Therefore, careful consideration of sample preparation, processing, and the amount of protein loading is necessary to achieve high-quality zymography results.

The optimized zymographic protocol presented here enables reliable detection of endopeptidase activities using gelatin or casein as substrates. Other substrates, such as collagen and fibronectin, can also be used to detect collagenase and fibronectinase activities, respectively. This approach facilitates a deeper understanding of metalloproteinase roles in ECM synthesis and degradation, particularly in matrix-related pathologies, including cancer and other tissue disorders. Zymography remains a widely used technique for visualizing ECM protein-degrading enzyme activity in plasma, urine, other body fluids, tissues, and cell culture samples.