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concentration range and the rate of precipitation of the remaining pair of
reagents is severely retarded. The phosphate anion is not functioning simply as
a calcium chelator, but it is performing some active primary function in the
precipitation process in partnership with the protein and calcium ions.
So, how successful are generic physical principles in explaining these phe-
nomena? For simple calcium-induced precipitation of a
s1
-casein, the simple
model of the polymer in poor solvent is a non-starter because the cation binds
to the protein rather than simply modifying solvent quality. But, for this
reaction, the classical DLVO model works very well (indeed, possibly better
than could realistically be hoped for), which points to a high degree of flexibility
and mobility of the casein molecules in solution. When phosphate is included in
the mixture, the mechanism of the precipitation process changes, and adherence
to the simple DLVO-type concept is lost. This is where the special chemistry of
the caseins emerges as a factor of major importance in understanding the
stability behaviour.
10.4 Casein Chemistry and its Role in Casein Micelle
Structure
The caseins are multi-phosphorylated, and the level of phosphorylation of the
individual casein monomers parallels their sensitivity to calcium-induced pre-
cipitation. In particular,
k
-casein, which generally carries only one phosphate
residue, is not precipitated by ionic calcium.
8
In the other caseins (a
s1
, a
s2
and
b), all of which are precipitated by Ca
21
, the majority of their phosphoserines
are clustered in groups of 3 or more. Figure 5 identifies the positions of these
cluster motifs in their respective sequences. It is a basic tenet of structural
biology - the Anfinsen hypothesis - that primary structure and biological
function are related. This hypothesis was enunciated to express the belief that
primary sequence structure defines secondary structure and conformation, but
a natural extension is that particular motifs in primary structure, particularly
those preserved across species and families of proteins, play a major role in the
biological function of those proteins. The phosphoserines of the caseins are
clustered for a purpose, and it is our contention, and also that of other
workers,
8-10
that the primary purpose of these motifs is to link within the
casein micelle with the calcium phosphate microcrystallites, also known as
'nanoclusters'. This interaction forms the basis of the cross-linking mechanism
in Holt's model of micellar assembly;
11
in fact, this is the only type of bonding
specifically mentioned in Holt's model, but it is only one of the ways of linking
casein molecules in the dual-binding model of Horne.
12
The other polymeriza-
tion linkage in the dual-binding model is via interaction of the segregated
hydrophobic regions found in all of the caseins. Both kinds of linkages allow
the development of a three-dimensional network extending through space, the
chains being terminated in the dual-binding model by
k
-casein, which thus
controls the casein micellar size.
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