Refining in vitro measurements of skin enzymes: catalase function and UVB effects, and regulation of ECM proteins breakdown by elastase and collagenase
Authors:   , Szczepanczyk Michal 
Published: November 2024

The skin is a highly complex organ with multiple functions, among which we focus on two: its enzymatic function and its role as a barrier. The stratum corneum acts as the primary barrier, protecting the organism from environmental threats, preventing water loss, and regulating permeability. Its homeostasis is maintained by an extensive enzymatic system. The skin is constantly exposed to both external and internal insults, which may lead to disorders and pathological changes often induced by oxidative stress. This state is characterized by an imbalance between the production of free radicals, primarily reactive oxygen species (ROS), and the skin’s ability to counteract or detoxify their harmful effects through neutralization by antioxidants. The antioxidant defence system in cutaneous tissues is closely interlinked and relies on both low molecular weight compounds, such as glutathione, vitamins, and urea, as well as antioxidant enzymes. Catalases are a crucial group of proteins for neutralizing ROS, with their primary role being the decomposition of hydrogen peroxide into harmless water and molecular oxygen. Hydrogen peroxide is a harmful byproduct of various biological processes, including cellular respiration and immune responses, and is thus ubiquitous. In this thesis, we investigated the antioxidant function of catalase by advancing the methodology for measuring catalase activity, building on standard techniques. We used one electrochemical method, relying on chronoamperometry with an oxygen electrode, and one spectrophotometric method, as presented in Paper I. Furthermore, in Paper II, we conducted a comprehensive study examining the effect of UVB light on the catalase activity and its structural properties. This study also investigates the effect of UVB radiation on the molecular organization of protein and lipid components of the SC. Furthermore, Paper III presents a fast and accurate chronoamperometric technique for detecting catalase activity in complex biological samples. Enzyme activity was measured in skin-related samples, including keratinocytes, stratum corneum with varying melanin levels, and defatted algae biomass (DAB) from Phaeodactylum tricornutum to evaluate this material as a potential redox- balancing agent that supports the skin's natural antioxidant barrier. Paper IV focuses on proteases, specifically collagenase and elastase, whose activity is crucial for the proper functioning of the dermis. These enzymes play a key role in the reorganization of the extracellular matrix (ECM), a complex network of macromolecules essential for maintaining tissue architecture, mediating cellular functions, and facilitating cell communication. Elevated protease activity has been linked to oxidative stress and is detrimental to the dermis, which is primarily composed of ECM. The upregulation of protease activity is also identified as a contributing factor to intrinsic skin aging and photoaging. In Paper IV, the aim was to develop substrate models of the two key structural ECM proteins, collagen and elastin, and to investigate the proteolytic activity of collagenase and elastase in a realistic manner. To achieve this, quartz crystal microbalance with dissipation monitoring and local surface plasmon resonance were employed to obtain complementary information on the rates of ECM proteins hydrolysis and changes in their viscoelastic properties. This research was conducted as part of the ComBine project, which involves training industrial doctoral students through collaborations between Malmö University and companies from the biomedical sector. Therefore, this thesis also includes unpublished results from research conducted in collaboration with Simris Group AB, aiming at evaluating defatted algae biomass as a potential source of topically applied bioactive compounds that contribute to skin protection and regeneration.