Isolation and characterisation of the core light-harvesting complex B875 and its subunit form, B820, from Rhodocyclus gelatinosus

We present a new method of preparation for Rhodocyclus gelatinosus light-harvesting complex B875 and its subunit form B820, without prior extraction of carotenoids from chromatophores. This method was based on partial dissociation of isolated B875 into B820 during hydrophobic chromatography in presence of ammonium sulphate. The two forms were then separated by gel chromatography in presence of this salt, which was required for stabilising B820. Carotenoid composition was determined for each form. B875 contained two hydroxyspheroidene molecules per bacteriochlorophyll dimer. Carotenoid content of B820 was about 5-times lower, and variable, indicating that most of this pigment was lost during the formation of B820. The apparent molecular mass of B820 antenna, determined by gel filtration, was 18-25 kDa. For B875 several values were obtained, depending on chromatographic conditions (protein and ammonium sulphate concentrations): > 600 kDa, 130 kDa and 35-40 kDa. These values may be inaccurate, because of the unknown contribution of detergent and possible interactions between the protein and the gel matrix. The results, however, indicated that B875 could present several states of aggregation, and that B820 has a smaller apparent molecular mass. Absorbance and CD spectra were measured and compared with spectra published for other species. We did not observe reconstitution of the original B875 form by increasing B820 concentration or decreasing ammonium sulphate concentration. However, another spectral form with absorbance maximum between 830-845 nm was observed, with a CD spectrum different in the infrared region from that of B820. A possible requirement of carotenoid for reassociation of B820 into B875 is discussed.