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 Geomyda 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 degrees C for 4 hr at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation constants are 7.2 x 10(-9) M for bovine trypsin and 5.5 x 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 that of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D004529:Egg White / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008970:Molecular Weight; D013381:Subtilisins / Q000037:antagonists & inhibitors*; D014302:Trinitrobenzenesulfonic Acid; D014361:Trypsin Inhibitors / Q000302:isolation & purification*; D014426:Turtles
D000596:Amino Acids; D014361:Trypsin Inhibitors; D014302:Trinitrobenzenesulfonic Acid; D013381:Subtilisins