The Binding of 1‐Anilino‐8‐naphthalene Sulfonate to the Hemocyanin of <i>Octopus vulgaris</i>

Ricchelli, Fernanda; Salvato, Benedetto

doi:10.1111/j.1432-1033.1979.tb12886.x

Cited by 6 publications

(4 citation statements)

References 33 publications

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“…Several proteins have been found to contain binding sites for the naphthalenesulphonate fluorescence probes (Stryer, 1968;Brandt & Gohlke, 1967). In particular, ANS was reported to be specifically bound at the active site of apohaemoglobin, apomyoglobin (Stryer, 1965) and apohaemocyanin (Ricchelli & Salvato, 1979). The changes in emissive properties of ANS in the presence of apo-tyrosinase are very similar to those reported previously and indicate that the probe is bound to the protein.…”

Section: Discussionsupporting

confidence: 79%

Fluorescence properties of Neurospora tyrosinase

Beltramini¹,

Lerch²

1982

Biochemical Journal

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Some structural properties of Neurospora tyrosinase have been studied by fluorescence spectroscopy. The emission spectra observed for oxy-, deoxy-, met- and apo-tyrosinase and the Co2+-substituted form are indicative of a protein containing buried tryptophan residues. By using acrylamide and iodide, part of the emission is quenched, indicating heterogeneity in the tryptophan environment. Upon binding of Cu2+ or Co2+ to apo-tyrosinase, a marked decrease of the tryptophan quantum yield is observed. A further decrease in emission intensity results from the binding of molecular O2 to the deoxy form. The fluorescent probe 8-anilinonaphthalene-1-sulphonate binds to tyrosinase only when the metal ions are removed. Reconstitution of apo-tyrosinase with Cu2+ completely displaces the probe, suggesting that 8-anilinonaphthalene-1-sulphonate binds to apo-tyrosinase at the active site. The fluorescence properties of Neurospora tyrosinase are compared with those of haemocyanin.

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Section: Discussionsupporting

confidence: 79%

Fluorescence properties of Neurospora tyrosinase

Beltramini¹,

Lerch²

1982

Biochemical Journal

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“…The Trp active site average distance in Octopus He was calculated according to the Forster equation with a number of energy acceptors, such as the copper-peroxide complex and carbon monoxide and ANS specifically bound to the active site in a 1:1 ratio (Alben et al, 1970;Yen Fager & Alben, 1972;Finazzi-Agro et al, 1982;Ricchelli & Salvato, 1979).…”

Section: Discussionmentioning

confidence: 99%

“…Full conversion to CO-Hc was checked by the constancy of the emission at 545 nm typical of the CO-Hc complex (Finazzi-Agro et al, 1982). The ANS-Hc complex was prepared from the apo form in 20 mM Tris-HCl buffer, pH 8.8, as described by Ricchelli and Salvato (1979). Chemicals were of the best grade commercially available and were used without further purification.…”

Section: Methodsmentioning

confidence: 99%

Emission quenching mechanisms in Octopus vulgaris hemocyanin: steady-state and time-resolved fluorescence studies

Ricchelli¹,

Beltramini²,

Flamigni³

et al. 1987

Biochemistry

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“…Two types of fluorescent labels can be used in order to detect conformational changes in proteins. One type, like l-anilinonaphthalene-8-sulfonic acid [21,, interacts with the protein hydrophobically. The other type binds to the protein covalently [37, 381. Tarantula haemocyanin was labelled with 22 different fluorescent dyes.…”

Section: Searching For the Appropriate Fluorescent Labelmentioning

confidence: 99%

Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole

Leidescher¹,

Decker²

1990

European Journal of Biochemistry

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Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole, the maximum wavelength A, , , of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for A, , , of the fully oxygenated haemocyanin were 531.5 nm (pH < 7.8) and 530.0 nm (pH > 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH < 7.2) and 535.2 nm (pH > 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S.Only four amino acids of the four-hexamer were labellcd with 7-chloro-4-nitrobenzo-2-oxa-I ,3-diazole. They were identified as lysine 484 on the purified peptide Leu-Arg-Lys-Phe-His-Arg. This amino acid is located on the surface of the four copies of subunit d.The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.Haemocyanins are the copper-containing respiratory proteins of the two largest invertebrate phyla, the Arthroooda and the Mollusca [l -31. They are oligomeric proteins of very high molecular mass, attaining 3.4 MDa in the horseshoe crab, Limulus polyphemus, and 9 MDa in the vineyard snail, Helix pomatia. Although amino acid sequence studies have revealed that one of the two copper-binding sites (copper B) has a common origin in molluscs and arthropods, the rest of the sequence, including the other copper-binding site (copper A) is clearly not identical [4]. There is also a profound difference in quaternary structure : molluscan haemocyanins form large cylindrical aggregates of about 30 nm in diameter and contain up to 160 oxygen-binding sites, while arthropodan haemocyanins occur as hexamers or multiples thereof.The arthropodan haemocyanins lend themselves particularly well to the study of allosteric interactions between subunits. All of these proteins contain integral multiples of a basic substructure containing six subunits. The smallest naturally occurring haemocyanins are singly hexameric and the largest consist of eight hexamers. These provide a series of aggregates graded by size which offer many possibilities for comparison. For example in the spider Cupiennius saki, single hexamers and two-hexamers occur concurrently, and in this case, the two-hexamers are significantly more cooperative than the single hexamers [5]. Cooperativity is sometimes very high, so that any effects on subunit interaction can be detected with ease.Here we will be concerned with the four-hexameric haemocyanin of the tarantula, Eurypelma californicum. This haemocyani...

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The Binding of 1‐Anilino‐8‐naphthalene Sulfonate to the Hemocyanin of Octopus vulgaris

Cited by 6 publications

References 33 publications

Fluorescence properties of Neurospora tyrosinase

Fluorescence properties of Neurospora tyrosinase

Emission quenching mechanisms in Octopus vulgaris hemocyanin: steady-state and time-resolved fluorescence studies

Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole

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