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.

Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya.

A Kunitz-type protease inhibitor purified from the latex of green papaya (Carica papaya) fruits was crystallized in the presence and absence of divalent metal ions. Crystal form I, which is devoid of divalent cations, diffracts to a resolution of 2.6 A and belongs to space group P3(1) or P3(2). This crystal form is a merohedral twin with two molecules in the asymmetric unit and unit-cell parameters a = b = 74.70, c = 78.97 A. Crystal form II, which was grown in the presence of Co2+, diffracts to a resolution of 1.7 A and belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 A.

Effects of mechanical wounding on Carica papaya cysteine endopeptidases accumulation and activity.

The mechanical wounding impact on the Carica papaya latex protein pattern was investigated by analyzing three latexes. A first one commercially available, a second harvested from unripe but fully grown fruits, both obtained from regularly tapped fruits. A third one was collected from similar fruits but wounded for the first time. The results demonstrated both quantitative and qualitative changes in the protein content and in the enzymatic activity. Repeated wounding results in either, accumulation or activation (or both of them) of papain, chymopapain and caricain. Furthermore, new cysteine protease activity was found to transiently accumulate in the latex collected from newly wounded fruits. The possible implication of this enzymatic material in the papaya cysteine endopeptidases pro-forms activation is discussed.