studies (16). So far, successful CD measurements on ad-In this paper spectroscopic data of a proteolytic enzyme adsorbed proteins in situ have been reported using dilute dissorbed on solid-liquid interfaces are discussed. The experiments persions of ultrafine silica particles for which absorption and consisted of time-resolved and steady-state fluorescence of tryptoscattering of light are minimized (17, 18).
phan residues and of circular dichroism (CD) which give informa-
In this paper spectroscopic experiments are described that tion on the tertiary and secondary state of the protein, respectively.
give information on the structure of protein molecules ad-
The spectroscopic properties are measured for the inhibited form sorbed on hydrophilic and hydrophobic dispersed particles.
of subtilisin 309 in situ on a hydrophilic silica surface and on a
The possibilities that are now offered by using a perfluorohydrophobic Teflon surface. The results are compared with those alkoxy fluorocarbon resin as a hydrophobic particle are most obtained for the protein in solution. In the case of fluorescence it is reasoned that the average excited-state lifetime and short internal promising.
rotation correlation times are indicative parameters for structural
The experimental approach was to determine CD and fluchanges in the protein. The internal rotation is superimposed on orescence (steady state and time-resolved) of the inhibited the rotation of the adsorbed protein which is immobile on the form of a proteolytic enzyme both in the dissolved and in fluorescence time scale. Fluorescence and CD both prove that the the adsorbed state. The enzyme is a bacterial serine proteinprotein alters its conformation when it adsorbs at low surface ase which is inhibited by phenylmethanesulfonyl fluoride coverage on hydrophobic Teflon particles. In that case the trypto-(PMSF). It will be further denoted PMS-Savinase. A prelimphan fluorescence lifetime is shortened which is accompanied by inary study has been reported previously (16). The conforan increase in the a-helix content. At monolayer coverage the mational state of the protein is studied more extensively in protein maintains its original structure, although minor changes this paper.
in fluorophore dynamics occur. On hydrophilic silica particles the results from both techniques do not point in the same direction. The fluorescence was not affected, irrespective of the surface occu-EXPERIMENTAL pation, while the CD experiments show a decrease in a-helix content at low surface coverage.