Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2031~
O.Z. 0050/41278
Two-dimensionally crystallized macromolecular fllms
The present invention relates to two-dimension-
ally crystallized macromolecular films which have been
produced with the assistance of a lipid monofilm carrying
5biotin units.
The formation of crystalline films of natural
macromolecules, espe~ially of proteins, i8 of particular
interest. Such films allow investigations of the struc-
ture of these substances, the possibility of analysis of
10structural relations between ~he molecules and, in some
cases, ~he development of novsl applications. However, it
is precisely the interesting protein~ which do not
naturally form crystals suitable for analysisO Hence
there has been no lack of attempts to generate crystal-
15lized protein ilms. Thus, Uzigiris ~ Kornberg (Nature
301 (1983) 125-129) have reported a method for forming
two-dimensional protein films on lipid films. This method
allows specific binding of proteins to natural or syn-
thetic lipid ligands present in the planar lipid film.
20However, despite the preparation of a num~ar of crystal-
lized protein films, the disadvantage was that a specific
lipid ligand, and thus a protein-antigen con~ugate, must
be available for each protein. In this connection the
formation of specific protein films, those of strep-
25tavidin, u~ing this technique was also proposed. For this
purpose, the lipid intended for ~he lipid film was
provided wi.th a biotin ligand so that, because of the
particularly high affinity of biotin for streptavidin~
the streptavidin molecule~ are able to attach themselves
30to the biotin ligands in the lipid film and form a two-
dimensionally crystallized streptavidin film in the
water/air transition zone. Direct inspection of such
films is pos-~ible using fluorescence microscopy, and the
crystalline nature can be determined by electron micro-
35scopy (Blankenburg et al., Biochemistry 28 (1989) 8214).
It is an object of the present in~ention to
prepare crystallized macromolecular films which have not
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been obtainable to date in a 3traightforward manner.
We have found that thi~ obiect i~ achieved by
two-dimensionally cry3tallized macromolecular film~ which
are compo~ed of a first lipid monofilm carrying biotin
units and of a second film which i9 composed of strepta-
vidin and attache~ itself to the biotin units and
cry~tallizes, and of a film which is composed of a
biotinylated macromolecula and is built up over the free
active centers of the streptavidin film.
~hu~, these crystallized macromolecular films are
composed of biotinylated macromolecules which are bound
to the streptavidin film. In another embodiment of the
films according to the invention the binding is achieved
indirectly via a low molecular weight or pol~meric biotin
ligand molecule. The crystalli~ation of the second
macromolecular film is induced both by the receptor
affinity binding and by the ordering of the streptavidin
film which i~ already present.
In a particular embodiment of the films according
to the invention the macromolecule~ are composed of
proteins.
The first lipid monofilm carrying the biotin
units characterizing the crystallized macromolecular
films according to the invention is generally composed of
the known amino functional lipids.
Examples o~ compounds which have proven particu-
larly advantageous ~or the purposes of the present
invention are N-biotinyl-S-~1,2-bis(octadecyloxycarbon-
yl)ekhyl)cysteine, N-biotinyl-S-(l-carboxy-2-~N,N-diocta-
decylaminocarbonyl)ethyl)cysteine, N-biotinoyl-S-1-phos-
phatidyl-3-glycero-~n-dimyristoyl, N,N-dioctadecyl-N'-
biotinylpropanediamine and N, N-dioctadecyl-biotinamide.
A streptavidin film i~ then attached to this
biotinylated lipid film via the specific receptor~.
Streptavidin is a protein which ha~ four identical
subunits, each of which i~ able to bind a biotin mole-
cule. The non-covalent binding between streptavidin and
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biotin is known to be one of the strongest; the X~ valus
is about 1015 mol-l. Thi~ at~achment of the streptavidin to
the biotin units of the lipid film result~ in an ordered,
two-dimensional film a~ i9 depicted by way of example in
Fiyure 1 with (A) = lipid, (B) = biotin and (C) = strep-
tavidin. Very large two-dimensional streptavidin regions
covering from 50 to 200 ~m are produced in this way. They
can be detected by means of fluorescence microscopy.
The attachmen~ of a biotinylated, water-soluble
macromolecule, in particular a biotinylated protein, to
the free attachment sites of the biotin surfaca is now
possible with the depicted surface of the two-dimension-
ally crystallized strep~avidin film. The water-soluble
macromolecules can be biotinylated poly(meth)acrylate,
polylysine, polyvinyl alcohol or dextran. They ~erve to
anchor another ligand molecule to the already crystall-
ized surface. Proteins, such as biotinylated monoclonal
antibodies or enzymes, are able to bind directly to the
streptavidin film and be built up in a surprisingly
straightforward manner to give crystalli~ed films.
Figures 2 and 3 show the films according to the inven-
tion, where X generally represents a macromolecule and
the ~quares 0 are intended to represent the protein.
The two-dimensionally crystallized macromolecular
film~ built up in this way can be obtained by binding a
water-soluble biotin ligand adapter molecule to the
streptavidin film and thus maklng new recognition struc-
tures available to another protein. It is also possible
to bind a water-soluble polymer with biotin and ligand
~tructure~ to the streptavidin film, in exactly the same
way as a biotinylated protein can be coupled to the
~treptavidin film. Details of the experimental procedure
are to be found, inter alia, in Blankenburg et al.,
Biochemi~try 28 (1989) 8214.
Detection of the film~ obtained in thi3 way i~
carried out in a straightforwaxd manner by direct inspec-
tion under the fluorescence microscope. The crystallinity
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of the re~ulting films can be checked by electron micro~-
copy after tran~fer by the Langmuir-Schafer technique.
The crystallized macromolecular film~ according
to the invention permit rapid and straightforward pre-
paration of samples for investigation of the structure ofthesa macromolecules which are not otherwise obtainable
in cry~tallized form. These films are also suitable for
monitoring enzymatic reactions when the protein type is
coupled with retention of its en7.ymatic activity to the
streptavidin film.
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