University of Groningen
Structure and function of hemocyanin
Konings, Wilhelmus Nicolaas
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1969
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Konings, W. N. (1969). Structure and function of hemocyanin Groningen: s.n.
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VIII. SUMMARY
Helix pomatia. Triangles
twentieth molecules. The
follows:
eths. No significance should
wn.
I
eight of a half molecule.
ment. Thus, the cylinder
fold axial symmetry.
61 postu­
1 Bruggen et al.
ourse of the association­
lc observations 149, howtentative, but we think
epresents a good starting
This thesis deals with the relationship between structure and function of
hemocyanin of the Roman snail, Helix pomatia. In Chapter II the properties
of arthropodan and molluscan hemocyanins have been summarized. The
differences between the hemocyanins of the two phyla support the suggestion
that they originated independently.
Hemocyanins are respiratory proteins, which bind one oxygen molecule
per two copper atoms. When oxygenated, hemocyanins are blue, when deoxy­
genated, they are colorless. The properties of oxy- and deoxyhemocyanin can
be explained by a model in which the valence of copper is not defined.
The methods used in the experiments are described in Chapter III.
The association-dissociation behavior of a-hemocyanin has been studied
by ultracentrifugal analysis (Chapter IV). Above pH 7 and below pH 5 whole
molecules (102 S) dissociate into half (64 S) and tenth (20 S) molecules. At
alkaline pH values further dissociation into twentieth (13 S) molecules is
observed. The dissociation is a cooperative process. The twentieth molecules,
with a mol.wt. of about 4.5 . 10 5 daltons, are the smallest biologically
active components that can be obtained.
The dissociation is reversible in the pH range 4 - 11. Reassociation is
induced by readjusting the pH to 5
7 or, at alkaline pH values, by the
addition of bivalent cations.
The association-dissociation behavior does not obey the rules valid for
simple equilibria, so there must be a certain heterogeneity in the bonds between
the structural units.
Whole molecules are dissociated into halves by hydrostatic pressure; this
results in an elevated baseline between the two components in the schlieren
pattern.
The experiments about the binding of oxygen and copper are described
in Chapter V. Reintroduction of monovalent copper into apo-a-hemocyanin
leads to complete return of the properties of native a-hemocyanin. The
presence of the copper atoms in hemocyanin is required for the reassociation
of tenth and twentieth molecules to whole and half molecules. In the recon­
stitution process the copper atoms are randomly distributed over the empty
binding sites of apo-a-hemocyanin. All copper binding sites are thus equivalent
and grouped in pairs; each pair forms a functional group. Upon storage for a
few months, part of the copper atoms in every molecule is oxidized to
57
Cu(II). This affects the oxygenation behavior.
The oxygenation curves of fresh a- and iJ-hemocyanin are non-hyperbolic
at alkaline pH values in the presence of calcium and magnesium ions. There
are marked differences in the influences of the pH on the oxygenation curves
between the two compounds.
Hemocyanin was succinylated in varying degrees with succinic anhydride
(Chapter VI). Not only the amino groups, and the hydroxyl groups of
tyrosine, serine and threonine were acylated, but also the hydroxyl groups of
the carbohydrate moiety.
Succinylation induces dissociation of hemocyanin. Even at very low degrees
of succinylation dissociation into twentieths occurred; more exhaustive succi­
nylation did not lead to smaller components.
Sedimentation and viscosity measurements, as well as electron microscopy,
showed that these succinylated components had undergone considerable un­
folding.
By progressive succinylation an increasing part of the copper can be
removed by EDTA; the residual copper remains fully functional. Since one
molecule of oxygen is bound per two atoms of copper this means that both
copper atoms of a functional group are labilized simultaneously.
The cooperativity of the functional groups which is observed in native
hemocyanin is completely lost by the introduction of succinyl groups.
On the basis of these results and on electron microscopic studies a tentative
model for the subunit organization of Helix pomatia hemocyanin is proposed
(Chapter VII).
S8