| We recently co-developed a method (Figure
11) using our confocal microscope that allows quantitative fluorescence
measurements of water drops containing one or more fluorescently
labeled proteins. We found that human serum albumin (HSA) and
fibrinogen behave very differently at the surface of a water drop than
within bulk water. Fibrinogen is known to polymerize in solution,
but it was not expected that at intermediate concentrations,
fluorescently labeled fibrinogen would make small aggregated domains at
the air-water interface (Figure 12). Using this confocal
technique, we can make ratiometric measurements of partitioning by
dye-labeled proteins between the bulk and surface phases. Fluorescence
recovery after photobleaching (FRAP) measurements confirm that
polymerized fibrinogen exchanges slowly with bulk protein, whereas HSA
localized to the air-water interface exchanges rapidly with bulk
protein. Interestingly, HSA was found to compete with fibrinogen for
the interface, and inhibit fibrinogen polymerization.
|
  Figure 11. (A) Representative image of Texas Red-labeled HSA (red, air-water interface on left) with measuring box (yellow). (B) Schematic showing calculation of surface intensity. Average bulk fluorescence is subtracted from interfacial intensity. |
 Figure 12. Fibrinogen microdomains (green) form at the air-water interface. |
| Our lab
is continuing to
explore these interesting protein phenomena. We have shown that
an FDA-approved surfactant can abolish protein adsorption to the
air-water interface, which may serve protective roles in human patients
undergoing cardiac surgery. In addition, we plan to generate
single-pair FRET-labeled proteins that have relevance to blood
clotting, and study their stability at the air-water interface compared
to bulk solution. Protein instability and conformational changes at
gas-liquid interfaces are relevant to many problems in biomedicine and
biotechnology. |