Surface-Enhanced Resonance Raman Scattering Spectroscopy as a Surface Topography Probe in Plant Photosynthetic Membranes

Strong resonance Raman (RR) and surface‐enhanced resonance Raman scattering (SERRS) signals from carotenoids were detected from thylakoid (stromal‐side out) vesicles and inside‐out (lumenal‐side out) vesicles isolated from spinach chloroplasts. The intensity of the signals from both types of membranes was comparable, indicating that plant carotenoids are exposed on or close to both surfaces or sides of the thylakoid membrane. This is in contrast to previous studies with bacterial photosynthetic membranes (Picorel et al., 1988, J. Biol. Chem. 263, 4374–4380; and 1990, Biochemistry29, 707–712) that show carotenoids selectively located on the cytoplasmic side. In addition_; strong RR and SERRS signals were detected from stacked and unstacked photosystem‐II‐enriched membrane fragments, demonstrating that carotenoids are also exposed on both surfaces of the appressed region of the thylakoid membrane. Antibodies against the photosystem (PS) II extrinsic proteins blocked SERRS signals from stacked PS II membrane fragments, but only partially affected the SERRS signals from unstacked membranes. The results indicate that these antibodies, which preferentially cover the surface of the original lumenalside of the appressed region, act as spacers between the membrane and SERRS electrode surfaces. The original stromal‐side of the appressed region is unaffected. These findings verify the distance sensitivity of the SERRS technique and underscore the above conclusion about the location of carotenoids in the appressed regions. Finally, SERRS signals are sensitive to membrane aging and storage temperature; caution is suggested to those applying SERRS spectroscopy to intact membrane systems.