The reaction of nitrite with the haemocyanin of the Roman snail (Helix pomatia).

The reaction of nitrite at pH 5.0-7.0 with the deoxyhaemocyanin of a mollusc, the Roman snail (Helix pomatia), yielded nitrosylhaemocyanin (CuIA.NO+ CuIIB), in contrast with the formation of methaemocyanin with the deoxyhaemocyanin of the crustacean Astacus leptodactylus (mud crayfish). With Helix haemocyanin 1 NO was thereby liberated per active site, as shown by m.s., as against 2 NO with Astacus haemocyanin. Helix nitrosylhaemocyanin was characterized in c.d. by the negative extremum at 336 nm (CuIA.NO+) and by the mononuclear e.p.r. signal at g = 2 (CuIIB). Binuclear e.p.r. signals have been observed after the addition of nitrite to methaemocyanins. With Astacus methaemocyanin, no further reaction occurred, whereas with Helix methaemocyanin the mononuclear e.p.r. signal, characteristic for nitrosylhaemocyanin gradually appeared. This formation of Helix nitrosylhaemocyanin implicates the binding, most likely on CuIIA, of a second nitrite besides a bridging nitrite, so that a dismutation into NO and NO2 can occur there. A further dismutation of NO2 yields nitrite and nitrate. The formation of the latter was demonstrated by Raman spectrometry. The reaction rate of Helix methaemocyanin with nitrite decreased with increasing pH according to the Henderson-Hasselbalch equation with a pKa value of 6.77, attributed to a mu-aquo bridging ligand, which can be exchanged for nitrite, in equilibrium with a mu-hydroxo ligand which cannot. These data also favour the formulation of the final reaction product as nitrosylhaemocyanin instead of semi-methaemocyanin, with or without bound nitrite.

The reaction of nitrogen monoxide with the haemocyanins of the crayfish Astacus leptodactylus and the snail Helix pomatia.

The rate of the reaction of Astacus leptodactylus methaemocyanin with NO follows the Henderson-Hasselbalch equation with a pKa of 5.85, suggesting that one imidazole ligand of Cu was exchanged for NO. The reaction is blocked by F- as a bridging ligand. The same imidazole residue might be responsible for the decomposition of nitrosylhaemocyanin, [Cu1NO+CuII], with an unlocated binding site for NO, into methaemocyanin and NO, as the rate increase with pH. NO could react preferentially with CuA of Helix pomatia methaemocyanin, CuA'IICuBII, as it possibly has only two histidine ligands instead of the three of CuA in Astacus haemocyanin. This difference might explain the higher formation rate and the much greater stability of Helix nitrosylhaemocyanin. The fast reaction is governed by a pKa of 6.80, probably of a bridging mu-aquo ligand. With F- or a mu-hydroxo bridging ligand a low reaction rate was still observed, in contrast with Astacus methaemocyanin. Helix nitrosylhaemocyanin was transformed by N3- into methaemocyanin with the liberation of N2 and N2O. This methaemocyanin could almost quantitatively be regenerated with H2O2. Helix nitrosylhaemocyanin was only partially regenerated by a direct treatment with H2O2 and almost quantitatively by HONH2 in a similar fairly fast reaction, followed by a much slower one.

The reaction of hydroxyurea with oxyhaemocyanin and methaemocyanin of the crayfish Astacus leptodactylus and the snail Helix pomatia.

The reaction of hydroxyurea with the oxyhaemocyanins of Astacus leptodactylus and Helix pomatia yielded methaemocyanins which could be regenerated with hydroxylamine. Hydroxyurea did not react with Astacus methaemocyanin, but quantitatively regenerated Helix methaemocyanin under N2. The reaction of hydroxyurea with Helix haemocyanin at pH 5.7 under air thus led to a steady state, with an oxyhaemocyanin/methaemocyanin ratio of 2.05:1.

The reaction of nitrite with the haemocyanin of Astacus leptodactylus.

The reaction of nitrite at pH 5.7 with deoxyhaemocyanin of Astacus leptodactylus yielded methaemocyanin in two one-electron steps, as nitrite was reduced to NO. This methaemocyanin could be almost fully regenerated by an anaerobic treatment with HONH2, in contrast with the methaemocyanin prepared with H2O2. A destruction of active sites on treating oxyhaemocyanin with HONH2 explains the partial regeneration of methaemocyanin under air, as traces of H2O2 are formed in the autoxidation of HONH2. The reaction rate of nitrite with deoxyhaemocyanin is almost 15 times that with oxyhaemocyanin. The slope of -1.0 for the logarithm of the pseudo-first-order rate constants plotted against pH indicates that HNO2 is the reacting species. Methaemocyanin was e.p.r.-undetectable, but a binuclear signal was observed at g = 2 on binding nitrite to methaemocyanin. This signal disappeared with a pKa of 6.50, suggesting that a mu-aquo bridging ligand, which can be replaced by nitrite, is deprotonated to a mu-hydroxo bridging ligand, which resists substitution by nitrite. The intensity of this triplet e.p.r. signal allowed the determination of the association constant of nitrite to the active site of Astacus methaemocyanin and yielded a value of 237 M-1 at pH 5.7. The interpretation by some authors of nitrosylhaemocyanin as a nitrite derivative of semimethaemocyanin is contradicted by this rapid reaction of nitrite with copper(I) in deoxyhaemocyanin and in semi-methaemocyanin and by the low binding constant of nitrite to the active site of methaemocyanin.

Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions.

For particulate suspensions and for solutions that scatter light measurably the total absorbance A generally contains contributions due to specific absorption (Aa) and scattering of light (As). The quantity As is closely related to the turbidity tau. In general, spectrophotometry of such systems requires proper modification of the spectrophotometer used in order to permit accurate determination of the absorbance A and of the derived quantities Aa and As. Apparent deviation from Beer's law in such systems is often due to inappropriate experimental technique. After a discussion of the parameters that determine the intensity of light scattered by solutes, an account is given of the experimental precautions to be taken for determination of the absorbance of light scattering suspensions and solutions and of techniques for correcting absorbance spectra for scattering of light. Measurement of the turbidity is briefly confronted with determination of the scattering ratio i90 degrees/Io and the impact of erroneous turbidity measurements on derived molecular parameters is discussed.

Complete amino-acid sequence of a functional unit from a molluscan hemocyanin (Helix pomatia).

From the beta c-hemocyanin (beta c-Hc) of the vineyard snail, Helix pomatia, the functional unit d (Mr approximately equal to 50,000-55,000) was isolated by limited proteolysis and gel chromatography. A small quantity of functional unit d was obtained intact, but the major part in the form of two peptides (Mr approximately equal to 43,000 and 10,000, respectively) connected by a disulfide bridge. After reduction and carboxymethylation, these were separated from each other and cleaved by conventional methods. The peptides were isolated by gel chromatography and HPLC, and sequenced manually or automatically. The complete sequence of Helix beta c-Hc d comprises 410 residues plus 3 residues at the N-terminus seemingly resulting from incomplete cleavage. There is apparently only one carbohydrate side-chain. Comparison of this gastropodan hemocyanin sequence to the partial sequence of a cephalopodan Hc C-terminal unit revealed sufficient identities to state that the functional units of molluscan hemocyanins have arisen by a series of gene duplications. On the other hand, there is practically no homology with arthropodan hemocyanins except for one section of 42 residues which is clearly homologous. This section corresponds to the "Copper B" site of Panulirus interruptus hemocyanin. It is also found in tyrosinases from Neurospora crassa, Streptomyces glaucescens, and mouse. In the N-terminal half of Helix beta c-Hc d there are other sections clearly homologous to the tyrosinases, but overall homology is limited. The second copper-binding site was not identified but must be completely distinct from the "Copper A" binding site of arthropodan hemocyanins. It is suggested that molluscan and arthropodan hemocyanins have evolved independently from a common ancestral mononuclear copper protein.

Isolation of mRNA from membrane-bound polysomes synthesizing Sepia officinalis haemocyanin in Xenopus laevis oocytes.

Isolation and translation in Xenopus laevis oocytes of the mRNAs from the nuclear pellet and the cytoplasmic supernatant of the branchial glands from Sepia officinalis, homogenized in the presence of vanadyl-ribonucleoside complexes, revealed that the membrane-bound polysomes for haemocyanin sedimented together with the nuclei. Centrifugation on a 15-50% sucrose gradient of these polysomes, released by treatment with Triton X-100, yielded a major peak of heavy ones. The messenger fraction, isolated from this heavy fraction, directed the synthesis of the large 390 000 Mr haemocyanin polypeptide chain in oocytes. A large mRNA on heavy membrane-bound polysomes thus synthesizes the whole haemocyanin chain of S. officinalis.

The structure of Artemia sp. haemoglobin. Cleavage of the native molecules into functional units by limited subtilisin digestion.

Limited subtilisin digestion of the high-Mr haemoglobin of the crustacean Artemia sp. results in a series of fragments that are multiples of Mr 16000. Properties such as amino acid composition, iron content, absorption and c.d. spectra of the 16000-Mr functional units strongly resemble those of the intact haemoglobin molecules. The 16000-Mr functional units can bind O2 in a non-co-operative way. They thus represent the structural units from which the globin chains are built up.

The regeneration with ascorbate of Helix pomatia methaemocyanin mediated by hydrogen peroxide.

The regeneration of the copper bands of H. pomatia haemocyanin proceeds much more slowly with an excess of ascorbate than with a slight excess of hydrogen peroxide. The regenerating agent with ascorbate is hydrogen perioxide, formed in its autoxidation at the air. This was concluded after regeneration experiments with ascorbate under strictly anaerobic conditions and at the air in the presence of catalase. The autoxidation of ascorbate was catalysed by Fe and Cu ions. In the presence of EDTA there is still metal catalysis, especially in slightly alkaline medium, due to the Fe(III)-EDTA complex. Addition of diethylenetriamine pentaacetate completely abolished the metal catalysis.

Stabilization of whole molecules of gastropodan haemocyanins by chlorhexidine.

Chlorhexidine diacetate 0.01% stabilized the whole molecules of Helix pomatia alpha-haemocyanin under dissociating conditions; in 1 M NaCl and in 1.42 M sucrose. Dissociation at low ionic strength (10 mM) of the haemocyanin of Pila leopoldvillensis was likewise prevented by chlorhexidine. After dissociation into half molecules of H. pomatia alpha-haemocyanin in 1.42 M sucrose and of P. leopoldvillensis haemocyanin by lowering the ionic strength, whole molecules were formed on introduction by dialysis of chlorhexidine diacetate to a concentration of 0.01%. This stabilization points to the binding of the two chlorophenyl groups in hydrophobic regions of the protein and an ensuing cross-linking of the half molecules of gastropodan haemocyanins. Chlorguanide, which almost corresponds to one half chlorhexidine, even at a concentration of 0.1%, only slightly stabilized the whole molecules.

Studies by small-angle X-ray scattering of the quaternary structure of the 24-S component of the haemocyanin of Astacus leptodactylus in solution.

The haemocyanin of Astacus leptodactylus was studied in several buffers at three pH values. The best stability and lowest mean deviation on repeating the measurements were found at pH 7.2 in a Tris-HCl buffer. The following molecular parameters were determined: radius of gyration 6.90 nm, radius of gyration of the cross-section 3.87 nm, maximum dimension 21.5 nm, relative molecular mass 854000, volume 1440 nm3, hydration 0.27 g H2O/g protein. The theoretical scattering curves of a large number of models were calculated to find one fitting these data and the experimental scattering curve. The model with the best agreement was compared with an electron micrograph.

Fragmentation of crystalline beta-haemocyanin of Helix pomatia with plasmin and trypsin. Location of the fragments in the polypeptide chain.

The action of plasmin on tenth molecules of beta C-haemocyanin of Helix pomatia at pH 8.2 yielded first a three-domain fragment P3 and a five-domain fragment P1 (present as a dimer at pH 8.2). Fragment P1 was further split by plasmin into a four-domain fragment P2 and a one-domain fragment P4 (also present as a dimer at pH 8.2). Trypsinolysis of P2 yielded T2 and fragment X, which was further split into T1C and T3. Fragments P3 and P4 corresponded respectively to the tryptic fragments T1A and T1B, also by their circular dichroic spectra. The determination of the N-terminal groups and the order of splitting allowed the location of the fragments in the polypeptide chain: P3 (a--c), P2 (d--g), P4 (h); T1A (a--c), T3 (d), T1C (e--f), T2 (g), T1B (h).

The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.

Studies by small-angle X-ray scattering of the quaternary structure of the beta-haemocyanin of Helix pomatia.

Helix pomatia beta-haemocyanin was studied in solution by small-angle X-ray scattering. The following molecular parameters were determined: molecular weight = 9.02 X 10(6), volume = 14000 nm3, radius of gyration = 18.4 nm, radius of the spherical subunits = 2.5 +/- 0.2 nm. With these data, and with information of dissociation products described in a former paper, a model of the molecule was built whose theoretical scattering curve showed good agreement with the experimental one. The model consists of 160 spherical subunits of a radius of 2.5 nm; 12 rings each built up of 10 spheres form the outer wall of a hollow cylinder; 20 subunits are situated at the inner side of each end.