Biomedical Engineering Reference
In-Depth Information
the Str-PE or Av-PE injected into the subphase solution with high affinity
( K a
10 15 ), producing a stability level nearly that of a covalent bond. Upon carefully inject-
ing the Str-PE or Av-PE into the subphase below the film, an altered low-pressure response
to film formation was observed for the LB film in the expanded state. This produced an
increased pressure compared with either the pure B-DPPE film or injecting the underiva-
tized PE into the subphase. This behavior suggests that the bulky derivatized PE, having
formed a complex with biotin, is somehow inserting itself into the expanded state of the LB
film during its formation. The PE protein is large (240,000 Da) and is disk shaped with
dimensions of 6.0 by 12.0 nm. In Figure 1.4 we present a schematic representation of how
this might happen (12). As the pressure increased above 15 mN/m, then all LB films exhib-
ited similar behavior. We believe this was due to the Str-PE, bound to the pendant biotin,
being pushed down into the subphase away from the B-DPPE molecules beginning to close
pack in the LB film. After forming the Str-PE immobilized LB monolayer film, it was trans-
ferred to the surface of a hydrophilic glass slide and allowed to dry, and the
fluorescence spectrum determined using a system that comprised an argon ion laser for exci-
tation, monochromator, and photocounter as shown schematically in Figure 1.5 (12). As
expected, the control films formed with either pure B-DPPE or PE injected under the B-
DPPE while forming the film exhibited no fluorescence, as shown in Figure 1.5. A normal
native fluorescence spectrum with emission maximum around 576 nm was observed for
both the LB films where either Str-PE or Av-PE (not shown) was injected into the subphase
to allow binding to biotin during film formation. Therefore, these phycobiliproteins retained
their optical properties through all of the binding and drying steps.
We have also taken an alternate film-based approach to the immobilization of fluores-
cent phycobiliproteins and other proteins. In this instance, we also utilized the
biotin-streptavidin interaction, but here the biotin was a pendant ligand covalently
1
3
2
525
535
545
555
565
585
595
605
615
625
635
576
(nm)
FIGURE 1.5
Fluorescence spectra of LB-monolayer films picked up onto glass substrates: (1) B-DPPE plus subphase injected
Str-PE protein conjugate; (2) B-DPPE plus subphase injected PE protein; (3) DPPE (phospholipid lacking biotin)
plus subphase injected Str-PE protein conjugate. Reprinted with permission from Samuelson, L.A., Miller, P.,
Galotti, D., Marx, K.A., Kumar, J., Tripathy, S.K., Kaplan, D.L. (1992). The Monomolecular Organization of a
Photodynamic Protein System through Specific Surface Recognition of Streptavidin by Biotinylated
Langmuir-Blodgett Films. Langmuir 8:604-608. Copyright (1992) American Chemical Society.
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