The haemocyanin of Pila leopoldvillensis—III. The dissociation studied by ultracentrifugation. Comparison with the α-haemocyanin of Helix pomatia

Elliott, Francis G.; Witters, Raphaël; Borginon, H.; Lontie, René

doi:10.1016/0305-0491(72)90326-4

Cited by 5 publications

(6 citation statements)

References 11 publications

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“…haemocyanins in terms of sensitivity of the 100S «-» 60S equilibrium to salt concentration has already been mentioned. However, it should also be pointed out that there are haemocyanins like that of Pila leopoldvillensis (Elliott et al 1972) in which the 100S particles cleave in low salt (< 15 mM) and are stabilized at high salt, the reverse of the behaviour of a-haemocyanins.…”

Section: (D) Association-dissociation Reactions Of Molluscan Haemocyamentioning

confidence: 99%

Haemocyanins

Holde

Miller

1982

Quart. Rev. Biophys.

256

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About ten years ago, one of the authors participated in a review of haemocyanin structure and function (van Holde & van Bruggen, 1971). At that time, it was possible to describe the field in terms of a limited amount of exciting new structural information, and a long list of unanswered questions. While the stoichiometry of oxygen binding was understood, virtually nothing was known about the active site. Even the oxidation state of the copper was a matter of conjecture. The size of the haemocyanin polypeptide chains was the subject of intense debate, with very little substantive knowledge available. While the haemocyanins were known to be allosteric proteins, there were virtually no experimental studies of oxygen binding on a level that could be meaningfully interpreted in terms of extant theories.

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Section: (D) Association-dissociation Reactions Of Molluscan Haemocyamentioning

confidence: 99%

Haemocyanins

Holde

Miller

1982

Quart. Rev. Biophys.

256

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“…3), and ultracentrifugation experiments indicate a fast decrease of the amount of halves present in the dissociation pattern in going from I = 0.05 t o 0.5 [3], halves seem not to be stable intermediates in the dissociation of wholes into tenths a t high ionic strengths. Even so a t I = 1.0 the existence of halves cannot completely be ruled out, preventing a rigorous quantitative evaluation.…”

Section: Ph -mentioning

confidence: 99%

“…The change in ionic strength has a completely different influence on the dissociation of Pila leopoldvillensis and of Helix pomatia ol-haemocyanin [3] : the former associates with the increase of ionic strength, whereas the latter dissociates under the same conditions. The association of Pila haemocyanin has been interpreted according to the Debye-Huckel theory for ionic interactions in terms of a reduction of the effective protein charge with increasing ionic strength [lo].…”

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confidence: 98%

“…I n this case the stock solution of haemocyanin (55 g/l) had a pH of 5.25 and 0.1 ionic strength (acetate 0.02 M ; KCl 0.08 M). According to Elliottt et al [3] the solutions were kept during 24 h a t 25 "C in order to obtain a complete equilibrium between the components. Before the light scattering measurements the solutions were cleared by centrifugation and decanted into the measuring cells.…”

Section: Solutionsmentioning

confidence: 99%

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Influence of Buffers, Protein Concentration and Ionic Strength upon the Dissociation of Pila Haemocyanin

Cox

Elliott

1973

European Journal of Biochemistry

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The study of the dissociation of Pila leopoldvillensis haemocyanin by means of light scattering has shown that the protein concentration has no influence upon the dissociation into halves and tenths.Different buffer ions were shown to have specific effects upon the dissociation of Pila haemocyanin: with maleate and phosphate the dissociation curves are shifted towards lower pH values; with imidazole and Tris the shift is in the opposite direction.Takening into account the high molecular weight of the protein, the binding of chloride ions, as determined by the AHp method, is very small. Therefore pure KC1 solutions rather than buffers were used in further work on the dissociation of haemocyanin.In view of these observations an attempt is made to interpret the influence of the pH and ionic strength upon the dissociation-association reaction according to a sequential loop model : the forward and back reaction proceed along opposite sequential pathways, each starting with a particular change of the ionisation state of the wholes, halves or tenths. According to this reaction scheme the ionisation constant of the sites responsible for the dissociation into halves is 6.96; and that for the dissociation of halves into tenths is 9.65. Halves reassociate in a perfect reversible way into wholes by a shift of pH from 6.35 to 5.15 as shown by zone electrophoresis [4] and electron microscopy [7]. The separation of two distinct components during ultracentrifugation or electrophoresis experiments should exclude however a direct interaction between wholes and halves [8]. This was confirmed in an extensive ultracentrifugation study on the dissociation of Helix pomatia haemocyanin [9]. Tenths too reassociate into wholes by lowering the pH; however, under the adopted experimental conditions, part of the reassociation products have dimensions exceeding those of wholes by factors as 3/2, 5/2 [7]. Within the pH stability regionThe change in ionic strength has a completely different influence on the dissociation of Pila leopoldvillensis and of Helix pomatia ol-haemocyanin [3] : the former associates with the increase of ionic strength, whereas the latter dissociates under the same conditions. The association of Pila haemocyanin has been interpreted according to the Debye-Huckel theory for ionic interactions in terms of a reduction of the effective protein charge with increasing ionic strength [lo]. The dissociation of Helix pomatia ol-haemocyanin with the increase of ionic strength has been attributed to the selective binding of chloride ions [9]. An indirect solvent effect of halogenide ions, as suggested by this author, seems rather improbable, since it should manifest itself also with other haemocyanins. I n Helix pomatia ol-haemocyanin the tendency for association, which would result from a reduction of the effective charge at high salt concentration, is completely suppressed by the dissociation induced by the chloride absorption.I n the present work the influence of different buffers on the dissociation of Pila leopoldvillensis ha...

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“…We report a somewhat lower molecular weight, 66,000, for subunits of Limulus hemocyanin. The minimum functional unit of molluscan hemocyanins is about 50,000 daltons, contains 2 copper atoms, but does not appear to correspond to the minimum molecular weight (7)(8)(9)(10)(11). Although low molecular weights (<100,000) have been reported for some molluscan hemocyanins (12)(13)(14)(15)(16) (4).…”

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confidence: 99%

Functional Differences in the Multiple Hemocyanins of the Horseshoe Crab, Limulus polyphemus L

Sullivan

Bonaventura

1974

Proc. Natl. Acad. Sci. U.S.A.

109

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Hemocyanin in the hemolymph of the horseshoe crab, Limulus polyphemus L., is a high-molecular-weight copper protein which binds oxygen cooperatively and shows a higher oxygen affinity at pH 7 than at pH 9. Treatment with EDTA (ethylenediaminetetraacetate) disaggregates the hemocyanin molecules and abolishes both the reverse Bohr effect and cooperative oxygen binding. Chloride ions interact with the EDTAtreated material and, in the presence of saturating amounts qf NaCl, a reverse Bohr effect is restored, but cooperativity is not. The EDTA-treated hemocyanin contains at least five electrophoretically distinct hemocyanins. These hemocyanins have similar molecular weights (about 66,000) but are functionally dissimilar.They have different oxygen affinities and different responses to chloride ions. The effect of chloride ions on unfractionated hemocyanin is due to pH-dependent chloride interactions with only two of the five hemocyanin components. The functional differences between the hemocyanin components may provide Limulus with a valuable respiratory flexibility in its interaction with the environment. The kinetics of oxygen combination and dissociation for the various hemocyanin preparations show that variations in the rate of oxygen dissociation are primarily responsible for the observed differences in oxygen affinity. The rate of oxygen dissociation varies 20-fold under conditions where the apparent rate of oxygen combination shows less than a 2-fold variation. Cooperative interactions in the untreated hemocyanin are most obvious in the "off" reaction, which increases in rate as successive oxygen molecules are released.

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The haemocyanin of Pila leopoldvillensis—III. The dissociation studied by ultracentrifugation. Comparison with the α-haemocyanin of Helix pomatia

Cited by 5 publications

References 11 publications

Haemocyanins

Haemocyanins

Influence of Buffers, Protein Concentration and Ionic Strength upon the Dissociation of Pila Haemocyanin

Functional Differences in the Multiple Hemocyanins of the Horseshoe Crab, Limulus polyphemus L

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