433 Environmental Toxicology and Chemistry, Vol. 17, No. 3, pp. 433–438, 1998  1998 SETAC Printed in the USA 0730-7268/98 $6.00 + .00 DIGESTIVE PROTEASES OF THE LUGWORM (ARENICOLA MARINA) INHIBITED BY CU FROM CONTAMINATED SEDIMENTS ZHEN CHEN and LAWRENCE M. MAYER* Darling Marine Center, University of Maine, Walpole, Maine 04573, USA (Received 26 March 1997; Accepted 30 June 1997) Abstract—We examined potential toxic effects of copper released from contaminated sediments during deposit feeding of the lugworm, Arenicola marina. Titration of Cu solution into gut fluids can result in decreases in protease activity if sufficient Cu is added. The effects of Cu on gut proteases were confirmed by incubation of gut fluids with Cu-contaminated harbor sediments. Monitoring of Cu titration into gut fluids shows that enzyme inhibition and quenching of gut protein fluorescence occur only when sufficient Cu has been added to allow inorganic Cu species to become abundant. This threshold level probably represents the exhaustion of strong binding sites that act as protection against enzyme inhibition. Thus, sediments contaminated with Cu may have inhibitory effects on digestive processes in lugworms. Keywords—Digestive fluids Lugworm Enzyme inhibition Sedimentary copper Deposit feeding INTRODUCTION Elevated concentrations of heavy metals, such as Hg, Cu, and Ag, in sediments can be toxic to benthic organisms by inhibiting enzyme systems or growth [1]. The inhibitory ac- tions on enzymes may include conformational changes due to metal binding [2], replacing original metals at catalytic sites [3], and blocking active catalytic sites [3]. In addition, sub- strates bound by metals may also lead to apparent losses of enzymatic activities [2]. For example, protease may not be able to bind and digest sedimentary substrates (i.e., peptides, proteins) loaded with metals (substrate tanning). On the other hand, metal–enzyme interactions have been successfully used to test the toxicity of sediment and soil samples [4], especially since the introduction of fluorophore-tagged substrates that dramatically enhance the sensitivity of enzyme assays. This method of enzyme assay has proved to be simple, sensitive, reproducible, and correlates well with other biologically based methods [5]. Marine deposit-feeding organisms rely heavily on secreted extracellular enzymes to hydrolyze sediment-bound organic materials. Because of the poor quality of sediment-based food, deposit feeders, such as the lugworm Arenicola marina, need to process large amounts of sediments continuously. Such a life style may make their digestive system vulnerable to sed- iment-bound pollutants. By incubating digestive fluids of two deposit feeders with contaminated sediments (i.e., in vitro di- gestion), we observed dramatic releases of copper and poly- cyclic aromatic hydrocarbons (PAHs) from the sediments [5]. The amounts of released pollutants were taken as a measure of their fractions in sediments bioavailable to the digestion of deposit feeders. Although it is operationally defined, the bio- availability of sedimentary Cu is a function of several factors, such as the sediments, species of organisms, and incubation time (i.e., gut retention time) [5]. The neutral gut pH of deposit feeders (Z. Chen and L.M. Mayer, unpublished data) suggests * To whom correspondence may be addressed (lmayer@maine.maine.edu). that H + is not likely responsible for solubilization of sedi- mentary metals. An alternative mechanism is complexation of sedimentary Cu by high concentrations of gut amino acids (e.g., up to 1 M) [5] (Z. Chen and L.M. Mayer, unpublished data). The objective of this study was to determine whether Cu- contaminated sediments causes damage to the digestive system of deposit feeders by monitoring the variation of protease ac- tivities in gut fluids under increasing Cu concentrations. We targeted lugworm protease in this study because protease is one of the major extracellular enzymes in the digestive system of the lugworm, and amino acid-based food items in sediments are important nutritional items for deposit feeders [6]. First we simulated the processes of Cu release from contaminated sediments by titration of Cu(NO 3 ) 2 solutions into a composite sample of gut fluid from multiple individuals. Then the pattern of Cu–protease interactions was further confirmed by addi- tional experiments, in which not only protease activities but also protein fluorescence and free cupric ion (Cu 2+ ) activities during the titrations were monitored to probe the mechanisms of protease–Cu interactions. Finally, we examined the effects of Cu released from contaminated sediments on lugworm pro- teases. MATERIALS AND METHODS Lugworms were collected from coastal Maine in summer 1995. The worms were kept in a flowing seawater table for a maximum of 2 d before being dissected for gut fluids. No detectable loss of enzymatic activities was found during this holding time (J. Judd, personal communication). To sample gut fluids, the midgut section was carefully exposed and taken out from a small cut on the body wall, and a pipette tip was used to penetrate and withdraw digestive fluids from this gut section. Digestive fluids from different individuals (normally 10) were pooled in order to achieve enough volume for incubation experiments. Particulate materials in gut fluids were removed by centrifugation at 8,000 g for 30 min. The gut fluids then were stored in plastic tubes at -80°C until experiments.