Original paper Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin N. Hellmann a, * , M. Paoli b , F. Giomi b,1 , M. Beltramini b, * a Institute for Molecular Biophysics, University of Mainz, Jakob-Welder-Weg 26, Mainz, Germany b Department of Biology, University of Padova, Viale G. Colombo 3, I-35131 Padova, Italy article info Article history: Received 8 September 2009 and in revised form 6 December 2009 Available online 4 January 2010 Keywords: Hemocyanin Crustacea Allosteric model Kinetics Oxygen binding abstract Hemocyanins from Crustacea usually are found as 1  6 or 2  6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of L-lactate. A par- ticular feature of this hemocyanin seems to be that L-lactate already enhances oxygen affinity under rest- ing conditions which possibly compensates the rather low intrinsic affinity observed in absence of L- lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration. Ó 2010 Elsevier Inc. All rights reserved. Introduction Mud shrimps spend most of their life in complex burrows, dug through littoral and sub-littoral soft sediments consisting of mud or muddy, fine sands. The oxygen circulation inside such a burrow is maintained exclusively through active water-pumping carried out by the crustacean. As shown for Callianassa truncata the oxygen concentration ranges from 5% to 40% of air-saturation inside the burrows, while oxygen is totally depleted in the surrounding sed- iments [1]. Mud shrimps exit these burrows for short periods in or- der to feed and reproduce [2,3]. Thus, in their daily life mud shrimps are exposed to water with different degrees of hypoxia. The lifestyle of these species suggests the evolution of a finely reg- ulated oxygen delivering system in order to maintain an efficient oxygen supply [4]. In some arthropods, namely in Crustacea and Chelicerata, oxy- gen is transported in the hemolymph by large, copper containing proteins, the hemocyanins. The hemocyanins of arthropods are multiples of hexameric assemblies of 75 kDa subunits. Depend- ing on the species hemocyanins are found as hexamers, dodeca- mers or multiples of dodecamers, displaying a rich set of quaternary structures of diverse levels of complexity and the pro- pensity to exhibit highly cooperative oxygen binding behavior [5]. Short-time regulation of oxygen binding behavior is accomplished by the ability of the protein to adopt different conformations with different binding affinities for oxygen. The relative amount of these conformations and consequently the mean oxygen binding affinity is regulated by effectors [6–9]. The extent of cooperativity and the oxygen binding affinity found for a given condition (temperature, pH, salt concentration) is highly species dependent and is assumed to reflect the needs imposed by the specific living conditions. In order to correlate the structural hierarchies with functional aspects, different models for cooperativity exist. Due to the large number of binding sites involved in case of hemocyanins mostly extensions of the concerted MWC model are used which is based on the assumption that a certain number of interacting binding sites are coupled (size of the allosteric unit) so that the whole structure can exist only in two conformations. These conforma- tions determine the binding behavior at low and high saturation levels, respectively. For hemocyanins only the simple hexamer was found to exhibit binding behavior which is in agreement with this simplest model, but even for hexamers the model failed in some cases [10]. Dodecameric hemocyanins can sometimes be de- scribed by a linear extension of the MWC-model where a third con- formation is postulated [11]. However, in most cases higher aggregation forms (2  6-, 4  6-, 8  6-meric) exhibit additional, hierarchical interactions. In this frame, it was suggested that struc- tural hierarchies should be reflected in hierarchies of the equilib- rium between different types of allosteric units, leading to the development of a theoretical model based on more than one level of cooperativity [12,13]. The nesting or nested MWC model is a hierarchical model where different allosteric units of the MWC- type are postulated and one level is embedded in the other [14,15]. 0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.12.025 * Corresponding authors. Fax: +49 6131 392 3557 (N. Hellmann), +39 049 8276300 (M. Beltramini). E-mail addresses: nhellman@uni-mainz.de (N. Hellmann), mar.paoli@gmail.com (M. Paoli), Folco.Giomi@awi.de (F. Giomi), beltmar@civ.bio.unipd.it (M. Beltramini). 1 Present address: Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany. Archives of Biochemistry and Biophysics 495 (2010) 112–121 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi