HYDROGEN PEROXIDE-SCAVENGING PROPERTIES OF SHEEP AIRWAY MUCUS

Reactive oxygen species released from luminal phagocytes in the airway can potentially injure the airway epithelium. Naturally occurring oxy gen radical scavengers must therefore exist to protect the epithelium. This study was designed to determine whether the high-molecular-weigh t fraction of normal sheep tracheal mucus has hydrogen peroxide (H2O2) -scavenging activity. Lyophilized mucus from 10 sheep was reconstitute d in phosphate-buffered saline (PBS) or Krebs-Henseleit buffer. H2O2 w as added to these mucus samples to a final concentration of 15 mu M, a nd the level of H2O2 remaining was measured over a 10 min period. From a zero-time level of 17 +/- 1.8 mu M (mean +/- SD), the H2O2 concentr ation fell within 10 min to 8 +/- 1.7 mu M in 0.05%; to 3.9 +/- 2.2 mu M in 0.1%; to 2.6 +/- 2.4 mu M in 0.2%; and to 1.2 +/- 1.5 mu M 0.4% mucus reconstituted in PBS. The results obtained in Krebs-Henseleit bu ffer were similar. The disappearance of H2O2 was not due to the transf ormation into hydroxyl radicals. Heat and acid denaturation and cleava ge of carbohydrate-free peptides from glycoproteins by pronase E treat ment abolished the scavenging potential. Fractionation of 0.4% mucus s amples according to molecular weight by gel filtration revealed that o nly one fraction with proteins of M(r) > 110 kD contained the active s cavenger. Polyacrylamide gel electrophoresis and lectin blotting with Ulex europaeus I (UEAI) showed that both the whole mucus and the activ ely scavenging gel filtration fraction contained a glycoprotein that c omigrated with a 205 kD molecular weight marker. With UEAI-coupled aga rose beads, the scavenging activity could be partially extracted from mucus; polyacrylamide gel electrophoresis revealed that the UEAI-posit ive glycoprotein was present in this extract. Immunoprecipitation expe riments and Western blots revealed that catalase was not responsible f or the measured activity; however, azide treatment abolished the activ ity. We conclude that sheep tracheal mucus contains at least one high- molecular-weight glycoprotein that actively scavenges H2O2 in a concen tration-dependent fashion and is possibly a secreted glycosylated pero xidase.