Hemocyanins

Hemocyanins (Hcl are oxygen-transport nonheme proteins I MW 10 lO ) which are found in the blood of some insects, crustaceans, and other invertebrates. One of the smallest He (MW 450,000) e.xiracted from spiny lobster Panulirus inierrupius consists of six subunits each containing two Cu atoms. Upon oxygenation, the deoxy form (Cu(I), colorless) turns to blue (Cu(ll), blue blood ) by binding one O2 molecule per two Cu atoms. 

Oxy-Hc extracted from C maghter (Pacific crab) and B. canaUculatum (channeled whelk) exhibit absorption bands near 570 and 490 nm. Freedman et measured the RR spectra of these compounds with 530.9 and 457.9 nm excitations. The results shown in Fig. V-15 clearly indicate that the bands near 

747cm are sensitive lo 02- 02 subsiituiion and must be assigned to the characteristic of the peroxo(0 ) type. Excitation profiles of the consist of iwo components and indicate that the absorption bands near 570 and 490 nm are due to Oz Cufll) charge transfer. These workers proposed 

X atom from a protein, possibly tyrosine. Recently, Gaykema et al. carried out X-ray analysis (3.2 A resolution) on colorless single crystals of He extracted from P. interruptus. This molecule consists of six subunits (MW 75,000), and each subunit is folded into three domains. The structure of the second domain in which two Cu atoms are located is shown in Fig. V-17. The Cu-Cu distance is 3.8 A, and each Cu atom is coordinated by three histidyl residues as suggested by Brown ei al. for the deoxy form. No evidence for a bridging protein ligand was found although it is not possible to rule out such a possibility from low-resolution X-ray analysis. 

Several groups of investigators prepared model compounds of He and demonstrated partial reversibility of oxygenation reactions of their compounds by electronic spectroscopyKarlin et al. are the first to observe the I fOo) (803cm ) of their model compound shown in Fig. V-18 using RR spectroscopy. 

There have been advances in the structural and physical properties of diiron complexes with dioxygen ligands. All of the well-defined structures have a, u-l,2-peroxo coordinated between two Fe(III) centers. These systems are not directly related to hemerythrin because of the mode of O2 bonding they are more relevant to the proposed structures in the active sites of ribonucleotide reductase (RNR) [5, 15] and MMOH [5] - two nonheme diiron-containing enzymes that activate dioxygen (vide infra). 

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Copper is one of the twenty-seven elements known to be essential to humans (69—72) (see Mineral nutrients). The daily recommended requirement for humans is 2.5—5.0 mg (73). Copper is probably second only to iron as an oxidation catalyst and oxygen carrier in humans (74). It is present in many proteins, such as hemocyanin [9013-32-3] galactose oxidase [9028-79-9] ceruloplasmin [9031 -37-2] dopamine -hydroxylase, monoamine oxidase [9001-66-5] superoxide dismutase [9054-89-17, and phenolase (75,76). Copper aids in photosynthesis and other oxidative processes in plants. 

Liquid chromatography/mass spectrometry Lower limit of detection Limit of detection Limit of quantitation Florseshoe crab hemocyanin Liquid scintillation counting Matrix-assisted laser desorption/ ionization mass spectrometry m -Maleimidobenzoy 1-A -Hydroxysuccinimide 1 -Cyclohexyl-3-(2-Morptiolino-ethyl)carbodiimide rnetlio-/ -Toluenesulfonate (same as CDI)... 

A similar effect has been observed for alamethicin I and II, hemocyanin, antiamoebin I and other substances. Great interest in the behaviour of these substances was aroused by the fact that they represent simple models for ion channels in nerve cells. 

Ross, P. K., and E. I. Solomon. 1991. An Electronic Structural Comparison of Cooper-Peroxide Complexes of Relevance to Hemocyanin and Tyrosinase Active Sites. J. Am. Chem. Soc. 113, 3246. 

Dissolve the protein to be modified at a concentration of 1-10 mg/ml in 0.1 M sodium phosphate, pH 7.4. NaCl may be added to this buffer if desired. For the modification of keyhole limpet hemocyanin (KLH Thermo Fisher) as described by Staros et al., 1986, include 0.9 M NaCl to maintain the solubility of this high-molecular-weight protein. If lower or higher concentrations of the protein are used, adjust the amounts of the other reactants as necessary to maintain the correct molar ratios. 

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH MW 4.5 X 105 to 1.3 X 107), BSA (MW 67,000), aminoethylated (or cationized) BSA (cBSA), thyroglobulin (MW 660,000), ovalbumin (OVA MW 43,000), and various toxoid proteins, including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine or mouse sera, tuberculin purified protein derivative, and synthetic polypeptides such as poly-L-lysine and poly-L-glutamic acid. 

Bartel, A., and Campbell, D. (1959) Some immunochemical differences between associated and dissociate hemocyanin. Arch. Biochem. Biophys. 82, 2332. 

Hersckovits, T. (1988) Recent aspects of the subunit organization and dissociation of hemocyanins. 

Senozan, N. et al. (1981) Hemocyanin of the giant keyhold limpet, Megathura crenulata. In Invertebrate Oxygen Binding Proteins Structure, Active Sites, andFunction (J. Lamy, and J. Lamy, eds.), pp. 703-717. Dekker, New York. 

Truchot, J.P. and F. Boitel. 1992. In vitro and in vivo effects of copper on hemocyanin-02 binding in the shore crab, Carcinus maenas. Comp. Biochem. Physiol. 103C 339-343. 

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