The enzyme carbonic anhydrase (CA) (EC 4.2.2.1.) is a catalyst of the reversible hydration of carbon dioxide to bicarbonate ions and protons (Maren, 1967). Carbonic anhydrase isozymes exhibit distinct differences in activity, tissue specificity and cellular localisation (Sly and Hu, 1995). Intraerythrocytic CA I and especially CA II are known to play an important role in the transport of CO 2 by blood (Klocke, 1988). Discussion continues as to whether CA IV, a membrane-bound carbonic anhydrase isozyme which, among other localizations, is present on the plasma face of vascular endothelium (Sender et al., 1998), significantly accelerates the efflux of CO 2 from tissue, e.g. from working skeletal muscle (Geers et al., 1997; Geers and Gros, 2000; Henry et al., 1997b) and, therefore, contributes to CO 2 transport within the body. Thus, it seems puzzling that both the plasma and serum of diverse vertebrate species including mammals, but not man and birds, have been shown to have an inhibitory effect on carbonic anhydrase (Booth, 1938; Haswell and Randall, 1976; Haswell et al., 1983; Henry et al., 1997a; Hill, 1986; Leiner et al., 1962; Maetz, 1956; Rispens et al., 1985). We aimed to identify any inhibitor of carbonic anhydrase present in the plasma of the European flounder Platichthys flesus and to determine its inhibitory properties and its molecular mass. A major purpose of the study was to investigate whether the serum of the flounder reduces the catalytic activity of carbonic anhydrase while the enzyme is enclosed in red blood cells. Materials and methods European flounder (Platichthys flesus) with a mean body mass of 200 g were caught in the Baltic Sea off Eckernfoerde, Germany. Live fish were brought to shore on the day of catch. Blood used for the preparation of serum and haemolysate was collected the same day, and red blood cell suspensions were prepared from blood samples taken from flounders that had been transported to our institute and kept in tanks filled with aerated artificial sea water. The continuously purified water had a temperature of 12–14 °C. The photoperiod was 12 h:12 h light:dark. The captive fish refused food. They were kept in the tanks for a maximum of 6 weeks before blood samples were taken. Fish were anaesthetised in a solution of 0.02 % (w/v) metacaine (3-aminobenzoic acid ethyl ester-methanesulphonate, Sigma) in sea water. Subsequently, each fish was given an intraperitoneal injection of 500 i.u. of heparin (Liquemin, Roche) and was then returned to the metacaine solution. After some minutes, animals were taken out again, sponged dry and bled by cutting off the caudal fin. One flounder yielded approximately 3 ml of blood. Blood was collected in 1.5 ml Eppendorf tubes containing 3003 The Journal of Experimental Biology 203, 3003–3009 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JEB2846 The blood serum of the European flounder Platichthys flesus strongly inhibits soluble erythrocytic carbonic anhydrase from the same species. The inhibition is of the uncompetitive type. Hence, the mechanism of the carbonic anhydrase inhibition is different from that of all other known carbonic anhydrase inhibitors. The serum showed no inhibitory effect on carbonic anhydrase from human and bovine red blood cells. By applying the 18 O exchange reaction, it could be demonstrated that the presence of the carbonic anhydrase inhibitor in the extracellular fluid has no effect on carbonic anhydrase in intact red blood cells. Thus, this carbonic anhydrase inhibitor seems to act only within the plasma space of the circulatory system. However, the carbonic anhydrase inhibitor does appear to reduce the bicarbonate permeability of flounder red cells to approximately one-quarter of normal levels as measured by the 18 O exchange reaction. The 28 kDa carbonic anhydrase inhibitor was isolated from the serum by gel filtration. The isolated inhibitor was detected in acrylamide gels as a single band representing a 7 kDa protein. The denaturing conditions used in electrophoresis presumably led to a dissociation of the native protein into subunits. Key words: blood CO2 transport, fish, uncompetitive enzyme inhibition, red blood cell, 18 O exchange reaction, European flounder, Platichthys flesus. Summary Introduction PROPERTIES OF A CARBONIC ANHYDRASE INHIBITOR PROTEIN IN FLOUNDER SERUM THOMAS PETERS*, FRAUKE PAPADOPOULOS, HANS-PETER KUBIS AND GEROLF GROS Zentrum Physiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany *e-mail: Peters.Thomas@mh-hannover.de Accepted 11 July; published on WWW 7 September 2000