Isolation and characterization of an aspartic protease from Salpichroa origanifolia fruits.
This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0-4.5 with hemoglobin and 5.5-6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin β-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.
Gabriela F
GF
Rocha
W David
WD
Obregon
Fernando
F
Munoz
M Gabriela
MG
Guevara
Graciela
G
Fernandez
Adriana M
AM
Rosso
Area de Quimica Biologica, Departamento de Ciencias Basicas, Universidad Nacional de Lujan, Lujan, Buenos Aires, Argentina. monicaparisiunlu@gmail.com.
Monica G
MG
Parisi
D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000302:isolation & purification*; D005638:Fruit / Q000737:chemistry; D007328:Insulin / Q000737:chemistry; D008969:Molecular Sequence Data; D019657:Solanaceae / Q000737:chemistry*
D007328:Insulin; D057055:Aspartic Acid Proteases