The antioxidant action of human extracellular fluids. Effect of human serum and its protein components on the inactivation of alpha 1-antiproteinase by hypochlorous acid and by hydrogen peroxide.

The elastase-inhibitory capacity of purified human alpha 1-antiproteinase is inactivated by low concentrations of the myeloperoxidase-derived oxidant hypochlorous acid, but much higher concentrations are required to inhibit the elastase-inhibitory capacity of serum samples. The protective effect of serum appears to be largely due to albumin. High concentrations of H2O2 also inactivate the elastase-inhibitory capacity of alpha 1-antiproteinase, by a mechanism not involving formation of hydroxyl radicals. Serum offers protection against H2O2 inactivation of alpha 1-antiproteinase. The relevance of these results to the tissue damage produced by activated phagocytes is discussed.

Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid. Implications for antioxidant protection in the inflamed rheumatoid joint.

Ascorbic acid, at physiological concentrations, can scavenge the myeloperoxidase-derived oxidant hypochlorous acid at rates sufficient to protect alpha 1-antiprotease against inactivation by this molecule. The rapid depletion of ascorbic acid at sites of inflammation, as in the inflamed rheumatoid joint, may therefore facilitate proteolytic damage.

Ozone effects on inhibitors of human neutrophil proteinases.

The effects of ozone on human alpha 1-proteinase inhibitor (A-1-PI), alpha 1-antichymotrypsin (A-1-Achy), bronchial leukocyte proteinase inhibitor (BLPI), and Eglin C were studied using in vitro exposures in phosphate-buffered solutions. Following ozone exposure, inhibitory activities against human neutrophil elastase (HNE) and/or cathepsin G (Cat G) were measured. Exposure of A-1-PI to 50 mol O3/mol protein resulted in a complete loss of HNE inhibitory activity, whereas A-1-Achy lost only 50% of its Cat G inhibitory activity and remained half active even after exposure to 250 mol of O3. At 40 mol O3/mol protein, BLPI lost 79% of its activity against HNE and 87% of its Cat G inhibitory activity. Eglin C, a leech-derived inhibitor, lost 81% of its HNE inhibitory activity and 92% of its ability to inhibit Cat G when exposed to 40 mol O3/mol. Amino acid analyses of ozone-exposed inhibitors showed destruction of Trp, Met, Tyr, and His with as little as 10 mol O3/mol protein, and higher levels of O3 resulted in more extensive oxidation of susceptible residues. The variable ozone susceptibility of the different amino acid residues in the four proteins indicated that oxidation was a function of protein structure, as well as the inherent susceptibility of particular amino acids. Exposure of A-1-PI and BLPI in the presence of the antioxidants, Trolox C (water soluble vitamin E) and ascorbic acid (vitamin C), showed that antioxidant vitamins may protect proteins from oxidative inactivation by ozone. Methionine-specific modification of BLPI reduced its HNE and Cat G inhibitory activities. Two moles of N-chlorosuccinimide per mole of BLPI methionine caused an 80% reduction in activity against Cat G, but only a 40% reduction in HNE inhibitory activity.

Detection of inhibitory inactive fraction of alpha-1-antitrypsin in human serum.

Pseudomonas aeruginosa elastase is not inhibited by human serum alpha-1-antitrypsin, it damages inhibitory activity of alpha-1-antitrypsin activity instead. A simple method, based on electrophoresis in urea containing polyacrylamide gel, is described for the separation of active part of the inhibitor from that inactivated by the bacterial enzyme.

The concentration of leukocyte elastase-alpha 1-proteinase inhibitor complex in bronchoalveolar lavage fluids from healthy human subjects.

Although alpha 1-proteinase inhibitor (alpha 1-antitrypsin) is widely thought to protect lung elastin against the elastolytic action of leukocyte elastase, there is only circumstantial evidence for such a protective role. We have demonstrated and quantified elastase-alpha 1-proteinase inhibitor complex in bronchoalveolar lavage fluids from healthy smokers and nonsmokers using a new enzyme-linked immunosorbent assay. The relative concentration of complex is 0.36 +/- 0.48 mmol/mol albumin in nonsmokers and 0.33 +/- 0.29 mmol/mol albumin in smokers. Less than 1% of lavage fluid alpha 1-proteinase inhibitor is complexed with elastase (0.31% in nonsmokers and 0.34% in smokers). This proportion is, however, much higher than in normal plasma where only approximately 0.006% of inhibitor is bound to elastase. Our data confirm that alpha 1-proteinase inhibitor efficiently acts as an antielastase barrier in the lower respiratory tract.

Inactivation of human plasma alpha 1-proteinase inhibitor by a metalloproteinase from Serratia marcescens.

The interaction of a Serratia marcescens metalloproteinase with human plasma alpha 1-proteinase inhibitor has been investigated. The enzyme was not inactivated by this inhibitor but, instead, converted the native plasma protein into an inactive form of decreased molecular weight. Amino terminal sequence analysis indicated that the interaction of the inhibitor and enzyme was at the reactive site of the inhibitor, with peptide-bond cleavage resulting in the inactivation. This process may be important in necrotic processes occurring during bacterial infiltration of host tissues.

Limited proteolysis by macrophage elastase inactivates human alpha 1-proteinase inhibitor.

Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. After degradation by macrophage elastase, alpha 1-proteinase inhibitor is no longer able to inhibit human granulocyte elastase, a serine proteinase implicated in the pathogenesis of emphysema. Macrophage elastase apparently does not degrade human granulocyte elastase-alpha 1-proteinase inhibitor complexes or release active granulocyte elastase from these complexes. The ability of macrophage elastase to degrade alpha 1-proteinase inhibitor is inhibited by EDTA and alpha 2-macroglobulin.

Possible mechanisms of emphysema in smokers. In vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants.

Freshly prepared, aqueous solutions of cigarette smoke suppressed the elastase-inhibitory capacity of human serum. Immunoelectrophoresis of mixtures of aqueous smoke solution, human serum, and pancreatic elastase showed decreased elastase/alpha1-proteinase inhibitor complexes and increased free, active protease. Phenolic antioxidants prevented the suppression of serum elastase-inhibition by cigarette smoke. By contrast, treatment of serum with model oxidants caused a similar suppression of elastase inhibition. These results suggested that emphysema in cigarette smokers might be due, in part, to the local suppression of alpha1-proteinase inhibitor in lung by oxidizing agents present in cigarette smoke.