In vivo binding of exogenous copper to haemolymph fractions of estuarine crab Scylla serrata (Forskal)

Abstract

In vivo binding of exogenous Cu to different haemolymph fractions (plasma, haemocyanin, clarified plasma and haemocytes) was quantitatively determined in specimens of the estuarine crab Scylla serrata (Forskal) injected with Cu as cupric chloride. As a prerequisite to this study, a suitable amount of Cu for injection was empirically selected by injecting various amounts of Cu (5.0, 7.5, 10.0, 12.5 and 15.0 μM; injection volume: 0.2, 0.3, 0.4, 0.5 and 0.6 ml, respectively) into the haemocoel of crabs and monitoring the consequent changes in haemolymph-Cu level at different time intervals post-injection. Based on the amount of injected Cu present in circulation as well as the reaching of an apparent asymptote of the haemolymph-Cu level after injection, an injection dose of 10 μM was found to be suitable to investigate in vivo binding of exogenous Cu to different haemolymph fractions. In the haemolymph of uninjected crabs, no free Cu is detectable, 86% of the total haemolymph Cu is found in association with plasma proteins and 13% is associated with haemocytes (haemocyte lysate supernatant). Analysis of protein in haemocyanin fraction isolated from plasma revealed that 83% of the protein in plasma is haemocyanin. When the crabs were injected with 10 μM Cu, no free Cu was detectable in any of the haemolymph fractions tested at any time post-injection, the first haemolymph sample was analysed at 30 min post-injection. At 2 h post-injection, the Cu concentration in haemocyanin and haemocyte-lysate supernatant increased significantly and accounted for 63 and 20% of the total injected Cu, respectively. These observations demonstrate that haemocyanin and haemocytes play a significant role in binding exogenous Cu. The Cu level in these fractions decreased thereafter and reached almost the pre-injection level within 4 h post-injection. Concomitantly at this hour of post-injection, the protein level in haemocyanin and haemocytes decreased significantly. These observations clearly indicate the disappearance of excess Cu from circulation along with the carrier proteins and suggest a probable role of haemocyanin and haemocytes in transport of exogenous heavy metals.