2-DIMENSIONAL CRYSTALLIZATION OF STREPTAVIDIN STUDIED BY QUANTITATIVEBREWSTER-ANGLE MICROSCOPY

Brewster angle microscopy (BAM) is extended to probe protein adsorptio n and aggregation processes at interfaces quantitatively. Since no p-p olarized light is reflected from the plain air-water interface under B rewster's angle, the grayscales of BAM images solely depend on the opt ical thicknesses of interfacial layers; photolabeling is not required. Effective refractive indices are calculated from the grayscales via F resnel's equations and are finally converted to relative protein surfa ce densities by the Maxwell-Garnett theory. It is shown here that the Maxwell-Garnett theory provides an accurate framework for the descript ion of a protein-water slab. Streptavidin binding to biotinylated mono layers at the air-water interface was chosen as a model system for an in situ study of the formation of two-dimensional streptavidin crystal s. H-shaped aggregates are seen with BAM. We identify these as two-dim ensional crystals since they are equal in size and shape to those typi cally observed in fluorescence microscopy which have been characterize d by electron diffraction. The difference in protein surface density b etween the streptavidin crystals and the noncrystalline surrounding is sufficient to provide for a rich contrast without the use of a fluore scence probe. A critical protein surface density equal to 75% of the c rystal density is found to be required for the crystals to form, a val ue that is independent of the protein bulk concentration. We have stud ied further the compressibility of the two phases. Whereas the density of the crystalline phase remains constant during compression, the non crystalline phase can be compressed to a surface density which exceeds that of the crystalline phase without initiating further crystal grow th. This leads to an inverted contrast in BAM; dark crystals are seen on a bright background. The protein density of the noncrystalline phas e can also exceed that of the crystalline phase upon the much slower p rocess of protein adsorption from sufficiently concentrated bulk solut ions. The nature of this two-dimensional phase transition is discussed .