Cu²⁺-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate by reptile sera

This study shows the EDTA-resistant, Ca²⁺ and Cu²⁺-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) compound in reptiles sera determined by spectrophotometry UV/Vis and chiral chromatography. Samples of ten reptile species were incubated with aliquot of 100 or 400 μM HDCP in presence of 100 or 300 μM Cu²⁺, or 2.5 mM Ca²⁺ or 5 mM EDTA at 37 °C for 30-60 min. The results shown an activator effect of Cu²⁺ on HDCP hydrolysis in freshwater turtles sera (Trachemys scripta, Chelydra serpentina and Macrochelys temminckii) because the levels of 2,5-dichlorophenol (DCP; product hydrolysis) were similar (∼37 μM DCP) to chicken serum (positive control group). The marine turtles (Chelonia mydas and Eretmochelys imbricata) and crocodiles (Crocodylusacutus and Crocodylus moreletii) showed ∼50% less HDCPase activity (13-17 μM DCP) compared to the HDCPase activity of the freshwater turtle species. Terrestrial reptile species (snakes and lizards) showed around 25% of activity (7-13 μM DCP) with both copper concentrations. These Cu²⁺-dependent hydrolysis were stereospecific to R(+)-HDCP (p˂0.05) in the three freshwater turtle species that showed similar hydrolysis to the chicken serum. However, the Ca²⁺ did not show a significant activating effect on the HDCPase activity (1-8 μM DCP) in any reptile serum. Their hydrolysis levels were very similar to those of EDTA-resistant activity. The present study demonstrates a Cu²⁺-dependent A-esterase (HDCPase) activity in turtles and points serum albumin as the cuproprotein responsible for this activity, reinforcing its N-terminal sequence (DAEH) as a catalytic center.