The early initiation of storage protein mobilization occurs through the Tubacin supplier active proteases, such as cysteine, serine, aspartyl, or metallo proteases deposited, during seed maturation, in protein bodies and vacuoles. The start of storage protein mobilization indicates that the mechanisms protecting storage proteins against degradation during middle and late maturation have been overcome. However, only few experiments have been performed with isolated protein bodies to show if the incubation under appropriate conditions leads to the breakdown of internal proteins that could be attributed to the action of these proteases [12, 13]. Thus, we were interested to verify the existence of proteases from C. echinata seeds, similar to the ones described, which could be involved in mobilization of seed reserves of this leguminous during early growth.

In the present work, we are reporting the purification and characterization of the first serine protease described in C. echinata seeds, named C. echinata serine protease (CeSP).2. Material and Methods2.1. Enzyme PurificationA crude enzyme preparation was obtained from C. echinata coatless dry seeds triturated and homogenized in 0.10MTris (purchased from Merck KGaA, Darmstadt, Germany) buffer pH 7.5 (1:20, w/v). The homogenate was centrifuged for 30min at 2,300g and the supernatant was subjected to an (NH4)2SO4 (purchased from Merck, Darmstadt, Germany) fractionation and allowed to stand overnight in cold. The resultant precipitated was recovered by centrifugation at the same conditions described above and dissolved in 50mM Na2HPO4/NaH2PO4 (purchased from Merck KGaA, Darmstadt, Germany) buffer, pH 7.

0. Proteolytic activity was examined in the hydrolysis of H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline Anacetrapib (H-D-Pro-Phe-Arg-pNA) (purchased from Chromogenix, Italy) that was used to monitor the purification procedure. The sample with proteolytic activity was applied to a HiTrap Phenyl column (purchased from GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA) equilibrated in 50mM phosphate buffer, pH 7.0, 1.0M (NH4)2SO4. Proteins were eluted in stepwise procedure with (NH4)2SO4 (0.95, 0.25, and 0.13M) at a flow rate of 2.0mL/min. The fractions with enzymatic activity were pooled, dialyzed against 20mM Tris buffer, pH 7.5, and applied to a Resource Q anion exchange column (purchased from GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA) equilibrated with 50mM Tris buffer, pH 7.5. Proteins were eluted with a NaCl (purchased from Merck KGaA, Darmstadt, Germany) gradient (0 to 0.50M) at a flow rate of 2.0mL/min.