In Vitro Exposure of Proteins to Ozone

Abstract

An in vitro system has been developed to expose proteins to ozone. The system is designed to deliver consistent and accurate levels of ozone over a range of concentrations (between 0.1 and ≥10 ppm) with extended exposure times (24 h or longer) in a humidified environment (100%). In the experiment presented in this article, ozone concentrations between 0.1 and 2.0 ppm were used. Ozone was generated by an electrical discharge ozonizer to ensure stability; it was continually monitored by an ultraviolet ozone analyzer and was precisely controlled by mass flow controllers, which gave reproducible results between runs. Humidity was closely regulated in the system to allow small amounts of protein solutions (50 μL or less) to be exposed without significant changes (<0.2%) in sample volume. The degree of surfactant protein-A (SP-A) oxidation by ozone was measured between runs to demonstrate the reproducibility of the system. A detailed description of the system is given, and protein oxidation detection methods and their limitations are discussed. Using these methods, we were able to assess oxidation of SP-A that apparently occurred prior to its isolation from the lung by bronchoalveolar lavage. This in vitro system allowed us to expose small amounts of protein to ozone in a simple, highly controlled, and reproducible manner.