Highly sensitive quantification of carbon black in lung burden through simplified proteinase K-mediated digestion

Accurate evaluation and quantification of nanomaterials are crucial in hazard and risk assessment. In this study, we tested simplified proteinase K (PK)-mediated digestion methods via a UVVis spectrophotometer for sensitive and accurate quantification of carbon black (CB) on the lung burden. We evaluated enzyme activity and digestion efficiency under different buffer conditions for lung burden digestion, focusing on the effects of varying surfactant and calcium chloride concentrations. The most effective conditions for PK characteristics with the model peptide substrate involved the use of Triton X-100, SDS, and CaCl₂. However, better results were observed for lung burden digestion when only SDS-containing media were used. Based on the influence of the interactions between lung burden tissue and PK, we improved the digestion efficiency and catalytic activity of PK. This led to 98 % recovery rates when 10 μg of PK was added to a buffer containing only the SDS surfactant within a 24-hour reaction time. These results demonstrate that the reaction media significantly affects the enzymatic reactions with different substrates. Proper conditions can be challenging to predict or engineer by solely studying the PK enzyme itself. Furthermore, this study provides potential fundamental features for future biomolecule extraction designs and clearance kinetics, utilizing protein digestive enzymatic strategies.