René Lontie

THE QUATERNARY STRUCTURE OF SEPIA-OFFICINALIS HEMOCYANIN

Abstract The haemocyanin (Hc) of Sepia officinalis is constituted of ten identical subunits ( M r about 390 000), which consist of eight functional (dioxygen-binding) units, designated by the letters a–h . A model is proposed for the quaternary structure of this HC, based on immuno-electron microscopy of whole molecules and on morphological studies of compact 1/5 molecules (consisting of two associated subunits). The immuno-electron microscopy was performed with IgG fractions containing antibodies specific for fragments abc, gh and de , respectively. Based on their general appearance, the immunocomplexes were divided into twelve types, whose occurernce was determined for the three IgG fractions. For each type of complex the location of the attachment sites of the IgGs was estimated in the cylindrical Hc molecule subdivided for this purpose into three regions (an upper, middle and lower third). The results indicate that functional units b to g form the wall of the cylindrical Hc molecule, while the two outer functional units of a and h are folded inside, thus forming a collar at the top and bottom of the cylinder. An experiment with a mixture of IgG fractions anti- abc and anti- gh showed that the ten subunits ( a–h ) lie parallel to each other, giving rise to an asymmetric molecule with, however, a symmetric appearance in the electron microscope.

The formation of Helix pomatia methaemocyanin accelerated by azide and fluoride

Haemocyanin, the oxygen-carrying pigment from the haemolymph of the mollusc Helix pornaria, shows two copper-oxygen absorption bands with a maximum at 346 and at 580 nm. These bands decrease slowly on storage [ I] . They reach about half their original value in one year and can be regenerated with hydrogen peroxide [2) and with hydrogen suiphide [3]. A considerable decrease of the copper bands after a few weeks, observed in the separation of cyand phaemocyanin in the presence of 1 M NaCl at pH 5.7, incited us to investigate the influence of halogenides. Similarly the abnormal bleaching of the colour of fresh haemocyanin by the addition of 3 mM sodium azide as a preservative led us to try the action of azide (C. Gielens, unpublished observation).

Relative molecular mass of the polypeptide chain of βc-haemocyanin of Helix pomatia and carbohydrate composition of the functional units

1. 1. Sedimentation equilibrium at pH 9.25 of the subunit (twentieth molecules) of βc-haemocyanin of Helix pomatia at protein concentrations below 0.6 mg/ml yielded a Mw value of ⋍4.5 × 105, in good agreement with the Mr value (9 × 106) of the whole molecule. 2. 2. The sum of the Mr values of the tryptic fragments from sedimentation equilibrium and of the eight functional units from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), amounted to 4.6 × 105. 3. 3. Carbohydrate is unevenly distributed over the functional units: g >b >e >a >h >d >f >c. On subtracting its weight contribution (8.25%) a Mr of ⋍4.2 × 105 was obtained for the polypeptide part of the subunit. 4. 4. The βc-haemocyanin subunit and some of its proteolytic fragments are proposed as markers for SDS-PAGE in the high-Mr range.

The preservation of haemocyanin under carbon monoxide.

The carbon monoxide combining capacity of haemocyanin has been in dispute for a long time. Root [I] concludes that binding occurs with Limulus haemocyanin from the solubility increase in serum of carbon monoxide by the presence of haemocyanin, and from the shift to the right of the oxygen dissociation curve. Rawlinson [2], on the contrary, does not find a difference in solubility of carbon monoxide between water and a solution’ of Palinurus vulgaris haemocyanin and consequently assumes no combination. Using r4C-labelled carbon monoxide, Vanneste and Mason [3] could demonstrate the binding of carbon monoxide by Cancer magister haemocyanin. We shall prove the formation of a compound with Helix pomatia haemwyanin by studying the competition between carbon monoxide and oxygen for the copper groups, and by measuring the solubility of carbon monoxide in a buffer with and without haemocyanin. During storage haemocyanin tiiidergoes an ageing process, consisting in a decrease of the absorbance at 346 nm [4] and a conversion of a sigmoidal to a hyperbolic oxygen dissociation curve. We shall try to prevent this transformation by storage under carbon monoxide, a method currently used for haemoglobin [5].

Presence of Only Seven Functional Units in the Polypeptide Chain of the Haemocyanin of the Cephalopod Octopus Vulgaris

The subunits of the haemocyanins (Hc’s) of gastropods (e.g. Helix pomatia, M r ≃ 450 000) and of decapodan cephalopods (e.g. Sepia officinalis, M r ≃ 390 000) are constituted of eight functional units (each containing a dioxygen-binding copper pair) with average M r of respectively ≃ 55 000 and ≃ 50 000 (1). In order to check if the Hc subunits of octopodan cephalopods are built in the same way, the subunits of Octopus vulgaris Hc were submitted to limited proteolysis.

The reaction of 4-(p-sulfophenylazo)-2-mercuriphenol with the thiol groups of proteins

1. 1.|A new water-soluble azomercurial, 4-(p-sulphenylazo)-2-mercuriphenol, has been prepared and tested as a thiol probe. 2. 2.|Upon reaction with the thiol groups of native proteins, the pK of the phenolic group of the reagent underwent a shift depending on the environment of the thiol group. An ovalbumin derivative, containing about 3 moles of azomercurial per mole of protein, showed two pK values for the phenolic group. 3. 3.|Using the large differences in visible absorption between bound and free dye, resulting from the pK shifts, a spectrophotometric thiol titration of β-lactoglobulin could be carried out. The thiol titration of bovine serum albumin was disturbed by adsorption of excess dye.

The fast reduction of Helix pomatia methaemocyanin with hydrogen sulphide

Oxygen binds reversibly to the copper of Helix pomatia haemocyanin in a ratio of 1 O,/ 2 Cu, giving rise to absorption bands at 346 and 580 nm [ 11. These bands show a continuous decrease when haemocyanin solutions are stored in air over a period of months [2]. This ageing can be prevented by storage of haemocyanin under carbon monoxide [3 J . Aged haemocyanin preparations can be regenerated by treatment with hydrogen peroxide, cysteine [4] or hydroxylamine [5]. It was clearly demonstrated that the very slow regeneration with cysteine is mediated by hydrogen peroxide [6]. This paper reports the fast regeneration of aged haemocyanin by treatment with small amounts of sulphide.

Gel chromatographic separation of the haemocyanins of Helix pomatia. Further electrophoretic and immunological characterization of the components

Abstract 1. 1. Chromatography on agarose gels at pH 5.5 in the presence of 1 M NaCI allowed to separate the total β-haemocyanin(β-Hc) of Helix pomatia from the α-haemocyanin (α-Hc). 2. 2. When crystalline β-Hc (β c -Hc) was first insolubilized by dialysis at pH 5.3 at low ionic strength the chromatographic separation yielded the soluble β-haemocyanin fraction (β 3 -Hc) and α-Hc. 3. 3. The amount of β c -Hc varied from one third to one half of β-Hc with the lowest value for non-hibernating snails. 4. 4. β 2 -Hc, in contrast to β c -Hc, did not differ from α-Hc in its electrophoretic and immunological properties.

The reversible reaction of cyanide with Helix pomatia haemocyanin

At low concentrations (< 150 μM) KCN does not remove copper from Helix pomatia haemocyanin. One cyanide ion replaces one oxygen molecule in the copper group, lowering the absorbance at 346 nm and the CD bands of haemocyanin between 300 and 800 nm.

Functional properties of the isolated domains of Helix pomatia beta c-hemocyanin.

Hemocyanins are large copper-containing proteins. They serve as oxygen carriers in many arthropods and molluscs and display allosteric behavior. The hemocyanin of the mollusc Helix pomatia has a relative molecular mass of 9 X 106. It 75% dissociated into half molecules in 1 M NaCl or KC1 in the stability region; this fraction was called a-hemocyanin. The fraction which does not dissociate under these conditions was called /I-hemocyanin [l] consisting of a &-component which crystallizes at pH 5.3, I 10 mM, and of a &-component which remains soluble [ 2,3]. On raising the pH the components dissociate into half molecules, which yield further tenth and twentieth molecules. The polypeptide chain of &-hemocyanin consists of 8 globular domains, like beads on a string, as can be seen from electron micrographs [4]. These functional domains, designated a-h, were isolated after limited proteolysis of tenth molecules with several enzymes [5]. They contain 2 copper atoms and have a relative molecular mass of 55 000 on average. Limited trypsinolysis of the undissociated cylindrical molecules removed the collar, leaving hollow cylinders which polymerized into tubes [6]. Oxygen binding by tubes proceeded with pronounced cooperativity. The slope of the high-affinity state was significantly <l, which indicated heterogeneity of the oxygen-binding sites. Similarly tenth molecules of fl,-hemocyanin of H. pomatia showed a Hill coefficient, h, of 0.9 under non-cooperative conditions at pH 8.1 [7]. Therefore the functional domains of wall fragments