The Reaction of Nitrogen Monoxide and of Nitrite with Deoxyhaemocyanin and Methaemocyanin of Helix pomatia

Abstract

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...

The reaction of nitrite with the haemocyanin of Astacus leptodactylus

“…For R = 100 and 200 almost the same final signal was obtained, indicating a saturation of the activesites with nitrite. In contrast with molluscan MetHc (Verplaetse et al, 1979), no NitrosylHc was formed.…”

Nitric oxide, a biological effector

“…Experimental settings: microwave frequency, 9.10GHz; field modulation frequency, 100kHz; field modulation amplitude, 1 G; microwave power, 10 mW. The same thing happens upon addition of NO to deoxyhemocyanin (Verplaetse et al 1979). B Same as A with a 5-fold expanded field scan around g=2, showing the superhyperfine structures.…”

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

“…Moreover MetHc yielded reversibly a nitrite derivative with a binuclear e.p.r. signal at g = 4 with seven hyperfine lines (Verplaetse et al, 1979).…”