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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.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and NzO 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(I1) 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.A green and stable nitrosylhaemocyanin has already been obtained in 1919 on treating anaerobically the deoxyhaemocyanin of Helix pomatia with nitrogen monoxide in slightly acid or in alkaline medium [I]. The addition of NO to deoxyhaemocyanin of Cancer magister gave a similar green derivative which showed a Cu(I1) electron paramagnetic resonance (EPR) signal [2]. The action of NO on the haemocyanin of H.pomatia at pH 5.7 yielded EPR signals of mononuclear Cu(I1) at g = 2 and binuclear Cu(I1) at g = 4 and at g = 2 [3]. Treatment with NO has to be carried out strictly anaerobically, as in a fast reaction with molecular oxygen NO yields N02, which in aqueous solution dismutates to nitrite and nitrate with the liberation of hydrogen ions. Thus the broad signal of binuclear Cu(I1) observed at g = 2 [3] was shown to be obtained when some dioxygen was present in the reaction mixture [4]. Nitrite in the presence of ascorbate yielded mainly a mononuclear Cu(I1) EPR signal at g = 2 [3,4].The possibilities provided by the Cu(I1) pair, present in H. pomatia methaemocyanin as indicated by the EPR signal at half field [5], encouraged us to reinvestigate the reaction of nitrite and NO with methaemocyanin and with deoxyhaemocyanin in order to elucidate the reaction steps. It will be shown that deoxyhaemocyanin yields methaemocyanin in a fast reaction with NO, as indicated by the appearance of an EPR signal at g = 4, as in [ 5 ] , and by the liberation Ahhrrviurions. CD, circular dichroism; EPR, electron paramagnetic resonance.of N 2 0 . Moreover, the presence of NO in the nitrosyl derivative of methaemocyanin will be demonstrated. MATERIALS AND METHODSH. pomatia haemocyanin was prepared by preparative ultracentrifugation of the haemolymph in a Spinco model L, rotor 30, for 2 h at 27500 rev./min at 4°C. The haemocyanin was dissolved in 0.1 M sodium acetate buffer, pH 5.0, and kept under carbon monoxide.Methaemocyanin was obtained by treatment with 25 mM sodium azide or 100 mM potassium fluoride at pH 5.0 and 37 "C for 2 days [6]. The solution was exhaustively dialysed for one week against several changes of 0.1 M acetate buffer, pH 5.0, in Visking dialysis membranes, especially treated [7] in order to remove traces of reducing agents able to regenerate haemocyanin. When needed, the haemocyanin solutions were concentrated by preparative ultracentrifugation in a Spinco...

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Recombinant human interferon gamma (IFN-gamma), produced in Escherichia coli, was selectively truncated at its C-terminus with chymotrypsin, clostripain or plasmin. The C-terminal amino acid residues of the three truncated IFN-gamma variants were identified as Phe136, Arg129 and Lys128, indicating the removal of 7, 14 and 15 amino acid residues from the full-length molecule. The absence of seven C-terminal residues did not influence the binding of IFN-gamma to its receptor. In contrast, the truncation of 14 residues resulted in a decrease in the Ka value to 1/24, as determined by surface plasmon resonance analysis. The removal of one additional amino acid residue from the C-terminal region of IFN-gamma led to a marked loss of receptor-binding capacity and biological activity. These observations demonstrate that Arg129 is an essential part of a functionally important C-terminal IFN-gamma sequence that is involved in receptor interaction.