Animal collection
Sea urchins were collected in the summer of 2010. Small (46 to 59 mm) and large (75 to 84 mm) S. purpuratus were collected near Taylor Island in Barkley Sound, British Columbia (48°49.572′ N, 125°11.823′ W). Small (38 to 50 mm) and large (158 to 165 mm) S. franciscanus were collected near Jackscrew Island, British Columbia (48°57′320″ N, 123°35.109′ W). Small (21 to 31 mm) and large (61 to 73 mm) L. variegatus were collected at Flatt’s Inlet, Bermuda (32°10.375′ N, 64°44.216′ W).
Collection of coelomic fluid
By analogy with arNOX in blood of humans, coelomic fluid was collected and immediately frozen after harvesting from urchins. The coelomic fluid was centrifuged at 6,000×g for 5 min to remove coelomocytes, and the resulting cell-free coelomic fluid (cfCF) was used to measure arNOX activity and protein amounts.
Assay of arNOX activity
arNOX activity was assayed from measurements of superoxide production based on a standard method, where the reduction of ferricytochrome c by superoxide was monitored from the increase in absorbance at 550 nm with reference at 540 nm (Butler et al. 1982). As a further check for the specificity of the arNOX activity, superoxide dismutase (SOD) was added near the end of the assay to ascertain that the rate returned to baseline. The assay consisted of 150 μl (2 mg/ml) oxidized ferricytochrome c solution and 50 μl of cfCF added to 2.5 ml assay buffer (0.15 M NaCl, 2.5 mM KCl, 1.5 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, and 7.5 mM glucose dissolved in deionized water, adjusted to pH 7.4, filtered and stored at 4°C). Rates were determined using a SLM Aminco DW-2000 spectrophotometer (SLM Instruments, Inc., Urbana, IL, USA) in the dual wavelength mode with continuous measurements over 1 min every 1.5 min. After 45 min, 60 μl (containing 60 units) SOD was added, and the assay was continued for an additional 45 min. An extinction coefficient for reduced cytochrome c of 19.1/mM/cm was used.
Determination of protein
Protein content was determined using the bicinchoninic acid method (Smith et al. 1985) (Pierce Technology Corporation, Iselin, NJ, USA). Standards were prepared with bovine serum albumin.
Statistical analyses
Means and standard deviations were analyzed for statistical significance using a two-tailed test. r2 values were determined by linear regression.