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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Reassembly experiments, involving isolated human apoproteins A-I and A-I1 and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-I1 molar ratios varying between 0 and 3. The apoproteins were incubated at 24 "C, 28 "C and 32 "C with either pure dimyristoylglycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 0, 5, 10, 15 mo1/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-I1 were quantified separately by immunonephelometry . The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence.The results suggest that apoprotein A-I1 has a greater affinity than apoprotein A-I for the phospholipidcholesterol vesicles and that apoprotein A-I1 is able to quantitatively displace apoprotein A-I from the lipidprotein complexes. The content of apoprotein A-I1 in the complexes increases proportionally to the concentration of apoprotein A-11 in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-I1 ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mo1/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-11.The physiological importance of apoprotein-phospholipid complexes has been emphasized by the observations of Hamilton et al. [I], indicating that discoidal particles consisting of phospholipids and apoproteins are generated as 'nascent' high-density lipoproteins (HDL) and by those of Forte et al.[2], who isolated discoidal HDL deprived of cholesterolesters in the plasma of patients deficient in phosphatidylcholine-cholesterol acyl transferase. During lypolysis and transfer of surface components from very low density lipoproteins (VLDL) and chylomicra to HDL, such intermediary structures are probably exchanged between lipoprotein classes [3].In view of the important role of these transient complexes in the synthesis and metabolism of the HDL particles, we have previously studied the composition, the structural properties and the thermodynamics of complexes generated between phosphatidylcholine-cholesterol mixtures and either apoprotein A-I or apoprotein A-I1 [4,5]. In this paper, we extend these studies to the mixed complexes containing phosphatidylcholine, cholesterol, apoproteins A-I and A-11, in an attempt to define the interrelationship between the apoproteins in the interaction with phospholipid and phospholipidcholesterol mixtures. These data should contribute to the understanding of the formation and the stabilization of circulating apoprotein-lipid complexes.Abbreviation...

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