Search results for "hemocyanin"
showing 10 items of 148 documents
Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthro…
1992
Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a …
Cooperative Transition in the Conformation of 24-Mer Tarantula Hemocyanin upon Oxygen Binding
2005
Hemocyanins are large respiratory proteins of arthropods and mollusks, which bind oxygen with very high cooperativity. Here, we investigated the relationship between oxygen binding and structural changes of the 24-mer tarantula hemocyanin. Oxygen binding of the hemocyanin was detected following the fluorescence intensity of the intrinsic tryptophans. Under the same conditions, structural changes were monitored by the non-covalently bound fluorescence probe Prodan (6-propionyl-2-(dimethylamino)-naphthalene), which is very sensitive to its surroundings. Upon oxygen binding of the hemocyanin a red shift of 5 nm in the emission maximum of the label was observed. A comparison of oxygen binding c…
Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2.
1997
Subunits of the two types of keyhole limpet hemocyanin (KLH1 and KLH2), purified by gel filtration chromatography and preparative polyacrylamide gel electrophoresis from Immucothel, have been used for macromolecular reassociation studies. In-vitro reassociation has been achieved with a standardized system using a Tris-saline stabilizing buffer at pH 7.4 containing 100 mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation has been monitored and the varying oligomeric forms of KLH1 and KLH2 produced have been studied by transmission electron microscopy, using specimens negatively stained with 5% ammonium molybdate containing 1% trehalose. Specimens ha…
Molecular characterisation and evolution of the hemocyanin from the European spiny lobster, Palinurus elephas.
2003
The hemocyanin of the European spiny lobster Palinurus elephas (synonym: Palinurus vulgaris) is a hexamer composed by four closely related but distinct subunits. We have obtained the full cDNA sequences of all four subunits, which cover 2275-2298 bp and encode for native polypeptides of 656 and 657 amino acids. The P. elephas hemocyanin subunits belong to the alpha-type of crustacean hemocyanins, whereas beta- and gamma-subunits are absent in this species. An unusual high ratio of non-synonymous versus synonymous nucleotide substitutions was observed, suggesting positive selection among subunits. Assuming a constant evolution rate, the P. elephas hemocyanin subunits emerged from a single he…
Identification, structure, and properties of hemocyanins from Diplopod myriapoda.
1999
Hemocyanins are copper-containing, respiratory proteins that occur in the hemolymph of many arthropod species. Here we report for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two immunologically distinct subunits in the 75-kDa range that are most likely arranged in a 36-mer (6 x 6) native molecule. It has a high oxygen affinity (P(50) = 4.7 torr) but low cooperativity (h = 1.3 +/- 0.2). Spirostreptus hemocyanin is structurally similar to the single known hemocyanin from the myriapod taxon,…
Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry.
2000
Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigme…
Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: a review of applications.
2002
Several representative examples are given of the successful application of negative staining across the holes of holey carbon support films using 5% (w/v) ammonium molybdate solution containing trehalose. The inclusion of 0.1% (w/v) trehalose is considered to be most satisfactory, although good data have also been obtained in the presence of 0.01 and 1.0% (w/v) trehalose. The examples given fall into the following groups: protein molecules in the absence of polyethylene glycol (PEG), protein molecules in the presence of PEG (Mr 1000), lipoproteins, lipids and membranes, filaments and tubules, viruses in the absence of PEG, viruses in the presence of PEG, aqueous polymer solutions, and final…
Hemocyanin from E. californicum encapsulated in silica gels: oxygen binding and conformational states.
2007
Cooperativity depends on the existence of equilibria among functionally distinct conformational states that are affected by homo and heterotropic effectors. In order to isolate the quaternary conformations of hemocyanin from E. californicum, the 24-meric giant protein was encapsulated in wet, nanoporous silica gels, either in the absence or presence of oxygen. The deoxy- and oxy-hemocyanin gels exhibit a p50 for oxygen of 11 and 2.5 torr, respectively, values in close agreement with those for hemocyanin in solution. The observed Hill coefficients are lower than unity, indicating a conformational heterogeneity within each locked conformational state, a finding in agreement with the assumptio…
Tryptophan quenching as linear sensor for oxygen binding of arthropod hemocyanins.
2008
Oxygen binding of hemocyanins results in an absorption band around 340nm and a strong quenching of the intrinsic tryptophan fluorescence. Our study analyses in detail the fluorescence quenching within two hemocyanins, a hexamer (Panulirus interruptus) and a 4 x 6-mer (Eurypelma californicum). Based on the comparison of calculated and measured transfer efficiencies we could show that: (1) For both hemocyanins FRET (fluorescence resonance energy transfer) is exclusively responsible for quenching of the tryptophan fluorescence upon oxygen binding. (2) Tryptophan quenching by FRET is independent of the oxy- or deoxy conformation of the protein. (3) The quenching takes place at the subunit level…
Cupredoxin-like domains in haemocyanins
2010
Haemocyanins are multimeric oxygen transport proteins, which bind oxygen to type 3 copper sites. Arthropod haemocyanins contain 75-kDa subunits, whereas molluscan haemocyanins contain 350–400-kDa subunits comprising seven or eight different 50 kDa FUs (functional units) designated FU-a to FU-h, each with an active site. FU-h possesses a tail of 100 amino acids not present in the other FUs. In the present study we show by X-ray crystallography that in FU-h of KLH1 (keyhole-limpet-haemocyanin isoform 1) the structure of the tail domain is cupredoxin-like but contains no copper. The copper-free domain 3 in arthropod haemocyanin subunits has also recently been reinterpreted as being cupredoxin-…