ADSORPTION-INDUCED CONFORMATIONAL-CHANGES IN THE SERINE PROTEINASE SAVINASE - A TRYPTOPHAN FLUORESCENCE AND CIRCULAR-DICHROISM STUDY

In this paper spectroscopic data of a proteolytic enzyme adsorbed on s olid-liquid interfaces are discussed. The experiments consisted of tim e-resolved and steady-state fluorescence of tryptophan residues and of circular dichroism (CD) which give information on the tertiary and se condary state of the protein, respectively. The spectroscopic properti es are measured for the inhibited form of subtilisin 309 in situ on a hydrophilic silica surface and on a hydrophobic Teflon surface. The re sults are compared with those obtained for the protein in solution. In the case of fluorescence it is reasoned that the average excited-stat e lifetime and short internal rotation correlation times are indicativ e parameters for structural changes in the protein. The internal rotat ion is superimposed on the rotation of the adsorbed protein which is i mmobile on the fluorescence time scale. Fluorescence and CD both prove that the protein alters its conformation when it adsorbs at low surfa ce coverage on hydrophobic Teflon particles. In that case the tryptoph an fluorescence lifetime is shortened which is accompanied by an incre ase in the cu-helix content. At monolayer coverage the protein maintai ns its original structure, although minor changes in fluorophore dynam ics occur. On hydrophilic silica particles the results from both techn iques do not point in the same direction. The fluorescence was not aff ected, irrespective of the surface occupation, while the CD experiment s show a decrease in alpha-helix content at low surface coverage. (C) 1997 Academic Press.