Role of Electrostatic Interactions on Molecular Self-Assembly of Protein + Phospholipid Films at the Air–Water Interface - Food Colloids: Self-Assembly and Material Science - page 229

For microscopic observation of the monolayer structure, the Brewster angle
microscope BAM 2 plus (NFT, Germany) was used as described elsewhere. 13,14
The BAM was positioned over the film balance. Measurements of surface
pressure, area, and grey level during monolayer compression were carried out
simultaneously. To measure the reflectivity of the monolayer from grey level
measurements a previous camera calibration was necessary. 13,14 The imaging
conditions were adjusted to optimize both image quality and quantitative
measurement of reflectivity; thus, as surface pressure increased, the shutter speed
was also increased.
AFM images of b-casein + DPPC monolayers were taken of Langmuir-
Blodgett films deposited onto hydrophilic mica substrates in the manner
described elsewhere. 9 Images were obtained in the constant force contact mode
in air or under redistilled n-butanol (Sigma) using an AFM manufactured by
East Coast Scientific Limited (Cambridge, UK). The imaging under n-butanol
yields reversible force-distance curves with no adhesive component under
retraction of the tip. Moreover, DPPC is soluble in n-butanol, a fact that can be
used advantageously in the analysis of images of DPPC + b-casein mixed films.
15.3 Results and Discussion
15.3.1 Effect of pH on Electrostatic Interactions
Figure 1 illustrates how pH affects the ionization of DPPC and b-casein in
monolayers at the air-water interface. At pH 5, some DPPC molecules will
be positively charged because the pH is close to the pK a value (3.8-4.0)
corresponding to dissociation of the first acid group. 16 Weak hydrogen bonds
between adjacent positively charged head-groups could lead to the formation of
a stable monolayer. At pH 7, the DPPC is zwitterionic which reduces the
formation of intermolecular hydrogen bonds. At pH 9, the DPPC monolayer
becomes completely ionized since the second acidic group dissociates (pK a ¼
8.0-8.5). 16 At this pH the incorporation of counter-ions into charged
phosphatidylcholine groups causes monolayer expansion by decreasing the
formation of intermolecular hydrogen bonds and increasing electrostatic
repulsion (see Figure 1A). 17 The b-casein molecule, a flexible linear polyelectro-
lyte, has a nonuniform distribution of hydrophilic and hydrophobic amino acid
residues. At pH 5, it will be close to having a net neutral charge, as this pH is
close to its pI (see Figure 1B). At pH 7, due to the amino acid content, b-casein
still has only a small net negative charge, which becomes much greater at pH 9.
However, the charge is located on the 40-50 terminal residues, with the rest of
the molecule remaining highly hydrophobic. Thus, although the force fields
that the protein molecules generate are predominantly electrostatic in nature,
the role of hydrophobic interactions must be significant for b-casein at the
interface, 18,19 these interactions being the main driving force for reversible self-
assembly in dilute solution. 20 The charged phosphoserine residues in b-casein
are considered to be repelled from the interface by electrostatic forces, leaving
the remainder of the hydrophobic groups strongly anchored to the interface. 21