Molecular exchange kinetics between a monolayer of antibody
molecules formed on the air−water interface
and the protein solution was studied by means of fluorescent labeling.
It was shown that there is no
inclusion of dissolved molecules in the previously formed monolayer
during even 6 h of exposure regardless
of monolayer surface density. The surface activity of IgG and
horseradish peroxidase molecules was
studied by means of surface compression isotherms, and the specific
biological activity of the monolayers
formed from these proteins was measured by enzyme and immunoassay
techniques. It was shown that
the surface activity of the proteins increases while specific
biological activity decreases with exposure of
the molecules on the water surface. Since the same effects were
caused by denaturing agents, we propose
that the surface activity of the proteins and the absence of
surface−volume exchange are due to partial
unfolding of the molecules which takes place on the water surface.
Two models of the partial unfolding
are discussed: complete denaturation of some part of the molecules
and partial unfolding of each molecule.
The process of surface denaturation was shown to be slow and
controllable. One can achieve a pronounced
increase of protein surface activity with low degradation of the
specific biological activity of the monolayer;
thus, it can be used in the practice of protein Langmuir film
deposition.
Monolayers of cytochrome P450scc and its complex with adrenodoxin
were formed by Langmuir techniques
and covalently immobilized on the solid substrates. The
orientation of hemeprotein molecules was studied
using polyclonal antibodies specific to the intact cytochrome P450scc
molecule and its tryptic fragments
F1 and F2, representing N- and C-terminal parts of the hemeprotein
molecule. Specific interactions of
the Langmuir films of cytochrome P450scc with adrenodoxin were
investigated, and the position of the
ferredoxin binding site at the hemeprotein molecule was identified.
It was shown that the molecular
orientation of the P450scc−AD (adrenodoxin) complex at the
water−air interface is dependent on the
surface density of the monolayer. The P450scc molecules do not
denature upon spreading on the water
surface. The formed monolayer can be transferred from the
air−water interface to the surface modified
with siloxane polymer and covalently immobilized without damage to the
structure. A model considering
the mode of orientation of cytochrome P450scc molecules on the
air−water interface in dependence on the
surface pressure is discussed.
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