Spectral analysis of chlorophyll-protein complexes from higher plant chloroplasts.
A spectral analysis of chlorophyll-protein complexes was carried out to gain information about the state of chlorophyll in vivo. A light-harvesting chlorophyll a/b protein complex and a Photosystem I complex were isolated from pea and from wheat chloroplasts by treatment with 0.5% Triton and centrifugation in a sucrose gradient. Resolution of absorption spectra (89K) of these fractions showed that their forms of chlorophyll were not altered by the isolation procedure. However, because the sum of the spectra of the fractions had a different shape from the chloroplast, it may be assumed that a third chlorophyll-protein complex was lost or changed in terms of the state of its chlorophyll. The spectrum of this missing chlorophyll was calculated and found to have a maximum near 683 nm. Circumstantial evidence indicates that this calculated spectrum may represent the native absorption of antenna chlorophyll a-protein of Photosystem II. The proportionality between the major absorbing forms of chlorophyll observed by curve analysis of different fractions suggests that the 660 and 678 nm forms may be the result of exciton interaction. The addition of a very small, narrow 675 nm band caused a very large improvement in fitting the spectrum of the antenna chlorophyll a/b protein with component bands, but not in Photosystem I spectra. A direct comparison of curve resolution with fourth derivative analysis shows the advantages of the former for studying the states of chlorophyll in vivo.