Preparation of crystalline Pseudomonas cytochrome c-551 and its general properties

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Abstract
Pseudomonas cytochrome c-551 was isolated and crystallized from P. aeruginosa, which was grown in submerged culture with nitrate as a terminal electron acceptor. The purification involves collection by H-form Duolite CS-101, chromatog-raphy on activated aluminium oxide, zone electrophoresis on a starch column and precipitation followed by crystallization from ammonium sulphate solution. After three recrystallizations, Pseudomonas cytochrome c-551 was a homogeneous protein according to electrophoretic and ultra-centrifugal criteria. The cytochrome shows absorption maxima at 551, 521, 416, 316, 290 and 280 m[mu] in the reduced form, and at 530, 409, 360, 290 and 280 m[mu] in the oxidized form. The molar extinction coefficient at 551 m[mu] ([alpha]-absorption maximum) is 28.3 x 106 mole-1cm.2 at pH 7.00. The cytochrome contains 16.2% of nitrogen and 0.69% of iron. The latter figure indicates a minimum molecular weight of 8100. Values of sedimentation constant, diffusion constant and partial specific volume at pH 7.0 (I 0.10) are S20 w, 1.34 x 10-13 cm. sec-1; D20.w, 14.8 x 10-7 cm.2 sec.-1; v, 0.710. The molecular weight calculated from these values is 7600 and the cytochrome thus contains one iron atom per molecule. The isoelectric point of the cytochrome is 4.70, and Eo (at pH 6.50 and at 20[degree]) is +0.286 v.