Absolute absorption and relative fluorescence excitation spectra of the five major chlorophyll-protein complexes from spinach thylakoid membranes

Abstract

The absolute absorption and relative fluorescence excitation spectra have been obtained for the five major chlorophyll-protein complexes from spinach chloroplasts, resolved by mild SDS-polyacrylamide gel electrophoresis. Electro-elution of the chlorophyll-protein complexes from the gels enabled their absolute absorption spectra to be determined. The mean molar extinction coefficient from 400 to 720 nm was independent of the type of protein complex and the chlorophyll a b ratio, averaging 2230 ± 40 m 2 per mol chlorophyll. The mean molar extinction coefficient was used to normalize the absorption spectra of the protein complexes while in the gel slices. The distribution of chlorophyll between the pigment complexes can be determined at 662 nm rather than from the mean of two scans at 650 nm and 675 nm. Recombining the absorption spectra from the pigment-protein complexes gave reasonable absolute agreement with the spectrum of spinach thylakoids. However, the solubilization led to (i) a shift in the red peak to shorter wavelengths by 4.5 nm, and (ii) a loss of carotenoids which also shifted the blue shoulder to shorter wavelengths. The absorption spectra for the two photosystems were predicted with phosphorylated (State 2) and nonphosphorylated (State 1) light-harvesting complexes. The 77 K fluorescence excitation spectra of the chlorophyll-protein complexes were measured in an attempt to assess the excitation spectra of PS I and PS II in terms of their chlorophyll-protein complexes. Due to the relative nature of fluorescence detection, the ratio of fluorescence excitation at 470 relative to that at 400 nm was used to compare the spectra. The discrepancies between the absorption and fluorescence spectra for the Photosystem I complexes meant that fluorescence emission was an insensitive method for assessing the degree of association of the light-harvesting chlorophyll a b- complex with each photosystem.