The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein.
The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive beta-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5-11.0) and temperature (up to 80 degrees C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.
Azarkan
Mohamed
M
University of Brussels, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808, route de Lennik, Bz-1070 Brussels, Belgium.
Dibiani
Rachid
R
Goormaghtigh
Erik
E
Raussens
Vincent
V
Baeyens-Volant
Danielle
D
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D001671:Biochemistry / Q000379:methods; D029441:Carica / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013025:Glycine max / Q000201:enzymology; D013696:Temperature; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D006023:Glycoproteins; C027132:1-anilino-8-naphthalenesulfonate; D014360:Trypsin Inhibitor, Kunitz Soybean; D004229:Dithiothreitol