MOLECULAR-ORIENTATION DISTRIBUTIONS IN PROTEIN FILMS .1. CYTOCHROME-CADSORBED TO SUBSTRATES OF VARIABLE SURFACE-CHEMISTRY

Molecular orientation in hydrated cytochrome c (cyt c) films formed by adsorption to substrates of differing surface chemistry was investiga ted. The orientation distribution of the heme groups in the protein fi lms was determined using a combination of two techniques: absorption l inear dichroism, measured in a planar integrated optical waveguide-att enuated total reflection geometry, and emission anisotropy, measured i n a total internal reflection fluorescence geometry. The mean heme til t angle and angular distribution about the mean were recovered using a Gaussian model for the orientation distribution. These data are the f irst orientation distribution measurements reported for protein film a ssemblies. The results show that a macroscopically ordered film of ads orbed cyt c molecules is produced when a single, high-affinity type of noncovalent binding occurs between the surface of the protein and the substrate surface. For example, electrostatic adsorption of the posit ively charged protein to the negatively charged head groups of a Langm uir-Blodgett film of arachidic acid produces a narrow orientation dist ribution. When multiple, competing adsorptive interactions are operati ve, which is the case when cyt c adsorbs to a clean glass surface, a r elatively disordered film is produced.