title: Reconsideration of phenoloxidase activity determination in white shrimp Litopenaeus vannamei.
abstract: Though phenoloxidase (PO) activity has been used as an important index in immunological research of crustaceans, methods for the determination of PO activity are not consistent even for the same species. Plasma, the major location of PO activity, should be the most reasonable sample, instead of hemocytes or serum, for the determination of PO activity of shrimp. The current study provided a thorough characterization and reconsideration for PO activity assay in the plasma of Litopenaeus vannamei. Results show that the final concentration of l-dihydroxyphenylalanine (l-DOPA) for PO activity assay should be no less than 1.5 mg ml(-1), and pH 6.6 should be used to maintain the stability of l-DOPA solution. This study provides direct evidence that PO activity is significantly inhibited by EDTA, and it is suggested to use EDTA-free anticoagulant in separating plasma for PO activity assay in future studies. Repeated measurements indicated that the assayed PO activities are significantly affected by preservation conditions, and plasma is quite unstable with spontaneous activation when put in ice or stored at -20 degrees C. Thus samples need to be measured immediately or preserved at -80 degrees C with assay as soon as possible after it is thawed, and should not be preserved for a second time for measuring PO activity.
authors.affiliation: Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, College of Life Science, South China Normal University, Guangzhou 510631, PR China.
authors.forename: Junwa
authors.initials: J
authors.lastname: Huang
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Anli
authors.initials: A
authors.lastname: Wang
mesh_terms: D000818:Animals; D004492:Edetic Acid; D006434:Hemocytes / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007980:Levodopa / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000032:analysis* / Q000378:metabolism; D033561:Penaeidae / Q000201:enzymology* / Q000276:immunology; D011309:Preservation, Biological / Q000379:methods
chemical_list: D007980:Levodopa; D004492:Edetic Acid; D014442:Monophenol Monooxygenase