Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.

Increases in tissue free amino acid levels in response to prolonged emersion in marine crabs: an ammonia-detoxifying process efficient in the intertidal Carcinus maenas but not in the subtidal Necora puber.

Carcinus maenas and Necora puber were exposed to air for 72 h and 18 h, respectively, at 18 °C. Changes in the free amino acid (FAA) content of their muscle, hepatopancreas and haemolymph were recorded during air-exposure and subsequent reimmersion. Muscle and hepatopancreas urate contents and haemolymph serum protein levels were also measured during emersion. In air-exposed C. maenas, the muscle FAA pool increased significantly within the first 24 h of emersion. This increase was due to an increase in the non-essential amino acid (NEAA) pool only; the essential amino acid (EAA) pool did not change. In haemolymph, the EAA pool decreased during the first 24 h of emersion, whereas the FAA and NEAA pools did not change. However, in this compartment, glutamine levels increased throughout the air-exposure period. No significant changes in FAA, NEAA and EAA contents of the hepatopancreas were observed during the 72 h emersion. In air-exposed N. puber, the FAA pools of muscle and hepatopancreas did not change, although changes in the levels of some amino acids were observed during the 18 h emersion period. In this species, large increases in both the NEAA and EAA pools in the haemolymph were recorded. High levels of urate were observed in the muscle and hepatopancreas of immersed N. puber, but no significant changes occurred during emersion. In contrast, immersed C. maenas exhibited low levels of urate in both compartments, and hepatopancreas urate levels increased slightly during emersion. Haemolymph protein content did not change in air-exposed N. puber, whereas it increased in the haemolymph of 72 h emersed C. maenas. The origin of newly formed NEAAs and their role in ammonia detoxification, particularly in C. maenas, which is able to regulate its internal ammonia levels during such a prolonged emersion, are discussed.