Abstract
The possible mechanism of casein aggregation and micelle buildup was studied in a new approach by letting α-casein adsorb from low concentration (0.1 mg·ml(-1)) solutions onto the charged surfaces of polyelectrolyte films. It was found that α-casein could adsorb onto both positively and negatively charged surfaces. However, only when its negative phosphoseryl clusters remained free, i.e. when it adsorbed onto a negative surface, could calcium phosphate (CaP) nanoclusters bind to the casein molecules. Once the CaP clusters were in place, step-by-step building of multilayered casein architectures became possible. The presence of CaP was essential; neither Ca(2+) nor phosphate could alone facilitate casein aggregation. Thus, it seems that CaP is the organizing motive in the casein micelle formation. Atomic force microscopy revealed that even a single adsorbed casein layer was composed of very small (in the range of tens of nanometers) spherical forms. The stiffness of the adsorbed casein layer largely increased in the presence of CaP. On this basis, we can imagine that casein micelles emerge according to the following scheme. The amphipathic casein monomers aggregate into oligomers via hydrophobic interactions even in the absence of CaP. Full scale, CaP-carrying micelles could materialize by interlocking these casein oligomers with CaP nanoclusters. Such a mechanism would not contradict former experimental results and could offer a synthesis between the submicelle and the block copolymer models of casein micelles.
Highlights
In milk, casein micelles serve the transport of calcium phosphate (CaP),2 the essential bone-building component, which is insoluble in water already at feeble concentrations
One of them says that micelles are created in two steps as follows: first, submicelles are formed, and these submicelles are organized to the final micelles; the second model says that the micelles are built by continuous incorporation of single molecules (block copolymers (5)), whose aggregation is governed by calcium phosphate nanoclusters, which are able to interact with several casein molecules (6)
Caseins are classified as members of the family of intrinsically unordered proteins (IUPs), which cannot be described by the characteristic proportion of ␣-helices, -sheets, turns, and unordered segments. (For a review on IUPs see Ref. 7.) by summarizing the results of several physical and spectroscopic studies, recently Farrell et al (8) concluded that caseins contain segments of the above mentioned classical secondary structures as well as a significant amount of polyproline II structure
Summary
The charged surface, governing the adsorption of casein onto the surface, was a “layer-by-layer” polyelectrolyte film built according to the method of Decher (10) from poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) having a negative or positive charge, respectively. Only a layer of about halfwavelength thickness of any material adsorbed onto the ATR crystal is sampled This is an ideal situation for polyelectrolyte films and adsorbed proteins of a thickness in the range of a few tens of nanometers. The difference absorption spectra calculated from the single beam spectra recorded before and after a given adsorption or chemical treatment contained all changes, i.e. the absorption spectrum of the adsorbed compound plus its effect on the underlying layers, accompanying the adsorption event. With this new approach, we introduced a model system, which is very close to that of the real conditions in the organisms. On the basis of our model system experiments, it seems that micelle formation (at least what concerns the ␣-caseins in it) might be a series of alternating calcium phosphate, and hydrophobic interactions mediated aggregation steps
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