Reactive oxygen species released from luminal phagocytes in the airway can potentially injure the airway epithelium. Naturally occurring oxygen radical scavengers must therefore exist to protect the epithelium. This study was designed to determine whether the high-molecular-weight fraction of normal sheep tracheal mucus has hydrogen peroxide (H2O2)-scavenging activity. Lyophilized mucus from 10 sheep was reconstituted in phosphate-buffered saline (PBS) or Krebs-Henseleit buffer. H2O2 was added to these mucus samples to a final concentration of 15 μM, and the level of H2O2 remaining was measured over a 10 min period. From a zero-time level of 17 ± 1.8 μM (mean ± SD), the H2O2 concentration fell within 10 min to 8 ± 1.7 μM in 0.05%; to 3.9 ± 2.2 μM in 0.1%; to 2.6 ± 2.4 μM in 0.2%; and to 1.2 ± 1.5 μM in 0.4% mucus reconstituted in PBS. The results obtained in Krebs- Henseleit buffer were similar. The disappearance of H2O2 was not due to the transformation into hydroxyl radicals. Heat and acid denaturation and cleavage of carbohydrate-free peptides from glycoproteins by pronase E treatment abolished the scavenging potential. Fractionation of 0.4% mucus samples according to molecular weight by gel filtration revealed that only one fraction with proteins of M(r) > 110 kD contained the active scavenger. Polyacrylamide gel electrophoresis and lectin blotting with Ulex europaeus I (UEAI) showed that both the whole mucus and the actively scavenging gel filtration fraction contained a glycoprotein that comigrated with a 205 kD molecular weight marker. With UEAI-coupled agarose beads, the scavenging activity could be partially extracted from mucus; polyacrylamide gel electrophoresis revealed that the UEAI-positive glycoprotein was present in this extract. Immunoprecipitation experiments and Western blots revealed that catalase was not responsible for the measured activity; however, azide treatment abolished the activity. We conclude that sheep tracheal mucus contains at least one high-molecular-weight glycoprotein that actively scavenges H2O2 in a concentration-dependent fashion and is possibly a secreted glycosylated peroxidase.
|Number of pages||8|
|Journal||American Journal of Respiratory and Critical Care Medicine|
|State||Published - Jan 1 1995|
ASJC Scopus subject areas
- Pulmonary and Respiratory Medicine