Purification And Biochemical Characterization Of Phenoloxidase From Brine Shrimp Artemia Sinica

Brine shrimp (Artemia sinica) is a genius of aquatic crustaceans in the phylum Arthropoda. It exists widely in all continents except Antarctica globally and mainly discovered in saltern in coastal provinces as well as inland saline lakes in China. Same as other invertebrates, brine shrimp lacks true immune antibodies and immune memory. A non-specific immune system is employed to deal with pathogen invasion in brine shrimp, in which phenoloxidase (PO) plays a pivotal role by catalyzing the melanization cascade. Isolation and characterization of PO from brine shrimp (A. sinica) (namely AsPO) will improve the elucidation of its innate immune pathway and mechanism and eventually benefit immune enhancer development aimed to elevate non-specific immune ability in invertebrates. However, as a model organism widely implicated in crustacean theoretical researches, report in the purification and biochemical characterization of AsPO is still absent. This study proposed to purify AsPO using gel filtration and ion-exchange chromatography, and characterize its biochemical and enzymatic properties systematically. It will facilitate to reveal the biochemical and enzymatic properties of AsPO and establish a foundation for further research in crustacean non-specific immune defense and regulatory mechanism.In this study, crude AsPO was extracted from adult brine shrimp (A. sinica)using tissue homogenation, high speed centrifugation, ammonium sulfate precipitation and dialysis. The combination of Superdex 200 gel-filtration and Q sepharose Fast Flow ion-exchanger were applied in AsPO purification. L-dihydroxyphenylalanine (L-DOPA) was used as the substrate in enzymatic activity assay. Near homogeneous AsPO was obtained by indicating a single band of 125.5 kDa on both reducing and non-reducing SDS-PAGE. Final purification fold in AsPO purification was 51.79-fold.The biochemical and enzymatic results indicated that the optimal pH for purified AsPO was 7.0 while optimal temperature was 50oC against L-DOPA. L-DOPA and catechol were identified as AsPO's specific substrates while its Km against L-DOPA and catechol were 4.3 mmol/L and 8.3 mmol/L respectively. Effects of oxidase inhibitors showed that PO activity was completely inhibited by ascorbic acid and sodium sulfite, significantly attenuated by citric acid, cysteine, benzoic acid, l-phenyl-2-thiourea and partially inhibited by thiourea. Combining the substrate specificity and inhibitory results, it can be concluded that AsPO is a catecholase-type PO. Besides, AsPO activity was inhibited by DETC and EDTA in a dose-dependent manner, inhibited by Zn²⁺ and Mg²⁺ but increased by Ca²⁺ and Cu²⁺, indicating AsPO is a metalloenzyme probably. Furthermore, the 20 mmol/L DETC-preinhibited AsPO activity could be restored specifically by 15 mmol/L Cu²⁺, implying that AsPO is a copper-containing metalloenzyme. All these demonstrated that AsPO is a copper-containing catecholase-type metalloenzyme.