Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white

Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11 700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100°C for 4 h at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation contants are 7.2·10^?9 M for bovine trypsin adn 5.5·10^?7 M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.     

Pulmonary hemodynamics and tissue damage after one lung infusion of Pseudomonas aeruginosa in sheep.

The relative roles of hematogenous mediators and direct bacterial toxicity due to phagocytosis by pulmonary intravascular macrophages were determined by selective bacterial infusion into the left pulmonary artery and comparison of right and left lungs at 24 h. Chronically instrumented sheep received 15-min pulmonary arterial infusions of live Pseudomonas aeruginosa (0.35-2.9 x 10(9), n = 6) or saline (n = 5). The saline group demonstrated stable cardiopulmonary function over time. Left lung blood flow, measured by Doppler flow probe, decreased 15 min into the bacterial infusion, with a concomitant sevenfold increase in left lung pulmonary vascular resistance index. The right lung pulmonary vascular resistance index doubled at 1 h, in association with increased plasma thromboxane B2 levels. An increase in cardiac index and decrease in systemic vascular resistance occurred at 12 h. The wet-to-dry weight ratio of the Pseudomonas-infused left lung was increased compared with that of the sham-infused lung. The tissue count of neutrophils in the lungs was doubled in both sides, but neutrophils on the left were more degranulated. The left lung tissue damage was caused by direct bacterial toxicity, including activation of phagocytic cells. Hematogenous mediators induced pulmonary and systemic hemodynamic changes and right lung neutrophil sequestration, but they did not damage the noninfused lung.