Haemocyanins

The modem era of biochemistry and molecular biology has been shaped not least by the isolation and characterization of individual molecules. Recently, however, more and more polyfunctional macromolecular complexes are being discovered, including nonrandomly codistributed membrane-bound proteins [41], These are made up of several individual proteins, which can assemble spontaneously, possibly in the presence of a lipid membrane or an element of the cytoskeleton [42] which are themselves supramolecular complexes. Some of these complexes, e.g. snail haemocyanin [4o], are merely assembled from a very large number of identical subunits vimses are much larger and more elaborate and we are still some way from understanding the processes controlling the assembly of the wonderfully intricate and beautiful stmctures responsible for the iridescent colours of butterflies and moths [44]. 

Ki Antagonist binding affinity Ki67 Nuclear membrane antigen KLH Keyhole limpet haemocyanin K , Michaelis constant KOS KOS strain of herpes simplex... 

Decker, H. and Rimke, T., Tarantula haemocyanin shows phenoloxidase activity, J. Biol. Chem., 273, 25889, 1998. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

In this method the keyhole limpet haemoglobin conjugate was prepared as follows Keyhole limpet haemocyanin (KLH, Calbiochem, La Jolia, CA) and bovine serum albumin (BSA, BDH Chemicals) were coupled to the adduct (2), derived from 6-bromohexanoic acid and monoquat (3), via a carbodiimide reaction, as reported previously by Niewola et al. [184], The resulting conjugates contained 662mol of Paraquat per mole of KLH and 15mol of Paraquat per mole of 6-bromohexanoic acid. The amount of Paraquat bound to the protein was determined by spectrophotometric dithionite assay for Paraquat and the protein concentration was established by a standard Lowry test. 

There has been enormous activity in the field of copper(I)-dioxygen chemistry in the last 25 years, with our information coming from both biochemical-biophysical studies and to a very important extent from coordination chemistry. This has resulted in the structural and spectroscopic characterization of a large number of copper dioxygen complexes, some of which are represented in Figure 14.2. The complex F, first characterized in a synthetic system was subsequently established to be present in oxy-haemocyanin, and is found in derivatives of tyrosinase and catechol oxidase, implying its involvement in aromatic hydroxylations in both enzymes and chemical systems. 

Catechol oxidase and tyrosinase are among the enzymes that have Type 3 dinuclear centres. However, the prototype of this class of proteins is the invertebrate oxygen transport protein, haemocyanin (Figure 14.5), for which structures of the oxy and deoxy forms have been determined at high resolution and confirm, as predicted from model compounds, that the... 

Figure 14.5 Copper-coordination sites in deoxy- and oxy-haemocyanin. (From Hatcher and Karlin, 2004. With kind permission of Springer Science and Business Media.)...

KLH Keyhole limpet haemocyanin, used for its excellent antigenic properties. It is used as a carrier protein in order to bestow immunogenicity in small haptens. 

The second class of dioxygen carriers is that of haemocyanins. These proteins, which contain a binuclear Cu(I) site (thus in the oxidized Cu(II) met form they belong to the so-called Type 3 copper proteins , which contain an EPR-silent dicopper active site), regulate dioxygen transport in the respiration of arthropods and molluscs. Figures 7 and 8 show... 

Figure 7 X-ray structure of the active site of the deoxygenated form of haemocyanin from Limulus polyphemus...

It is noted that in the deoxygenated form of haemocyanin from Panulirus interruptus the Cu/Cu separation is notably shorter 3.54 A.10... 

As previously discussed, the binuclear Cu(I) haemocyanins constitute another important class of dioxygen carriers. The first model compound whose structure in the oxygenated form resembles most closely that of oxyhaemocyanin is the dicopper(II) complex with the tris(3,5-diisopro-pylpyrazolyl)borate ligand shown in Figure 26.29... 

Copper is usually present in food at levels of 1 to 2 pg per g, the higher levels are found in animal livers. The highest levels of copper are present in shellfish, this is because copper is a component of their blood pigment, haemocyanin. 

Hati, S. and Datta, D. (1995). Nature of the active sites in haemocyanin and iron-rich hydgrogenases The bond valence sum approach. J. Chem. Soc. Dalton Trans. 1177-82. 

Lontie, R., Gielens, C. Molluscan and arthropodan haemocyanins. In Metalloproteins. Structure, Molecular Function and Clinical Aspects (Weser, U., ed.), Stuttgart-New York, Thieme, 1979, pp. 62-72... 

Protein molecules Egg albumin Serum albumin Serum globulin Haemocyanin 4.0 nm 5.6 nm 6.3 nm 22.0 nm... 

Bushara, H.O., Bashir, M.E., Malik, K.H., Mukhtar, M.M., Trottein, F., Capron, A. and Taylor, M.G. (1993) Suppression of Schistosoma bovis egg production in cattle by vaccination with either glutathione S-transferase or keyhole limpet haemocyanin. Parasite Immunology 15, 383-390. 

Xu, S., Shi, F., Shen, W., Lin, J., Wang, Y., Ye, P., Tian, E., Qian, C., Lin, B., Shi, Y. and Zhang, Z. (1995) Vaccination of sheep against Schistosoma japonicum with either glutathione S-transferase, keyhole limpet haemocyanin or the freeze/thaw schistosomula/BCG vaccine. Veterinary Parasitology 58, 301-312. 

Alves-Brito, C.F., Simpson, A.J.C., Bahia-Oliveira, L.M.G., Rabello, A.L.T., Rocha, R.S., Lambertucci, J.R., Gazzinelli, G., Katz, N. and Correa-Oliveira, R. (1992) Analysis of anti-keyhole limpet haemocyanin antibody in Brazilians supports its use for the diagnosis of acute schistosomiasis mansoni. Transactions of the Royal Society of Tropical Medicine and Idygiene 86, 53-56. 

Hamilton, J.V., Chiodini, P.L., Fallon, P.G. and Doenhoff, M.J. (1999) Periodate-sensitive immunological cross-reactivity between keyhole limpet haemocyanin (KFH) and serodiagnostic Schistosoma mansoni egg antigens. Parasitology 11 8, 83-90. 

Wuhrer, M., Dennis, R.D., Doenhoff, M.J. and Geyer, R. (2000a) A fucose-containing epitope is shared by keyhole limpet haemocyanin and Schistosoma mansoni glycosphingolipids. Molecular and Biochemical Parasitology 1 1 0, 237-246. 

Byrt, P. Ada, G.L. (1969). An in vitro reaction between labelled flagellin or haemocyanin and lymphocyte-like cells from normal animals. Immunology 17,503-516. 

Figure 2.7 Sedimentation of a monodispersed sample of Limulus haemocyanin measured by the Philpot-Svenson schlieren method (18 000 rev min l)32 (By courtesy of the American Chemical Society)...

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