NONHEME IRON PROTEINS 



319 



300 



400 500 600 700 

 WAVELENGTH (m;j) 



Fig. 2. Difference spectrum (oxidized-minus-reduced) 

 of Cliro»?afii())i iron protein. The sample cuvette con- 

 tained 0.34 mg/ml oxidized iron protein in 50 mM phos- 

 phate buffer, pH 7; the reference cuvette contained the 

 same mixture plus approximately 1 mg mercaptoethanol 

 added to reduce the protein. 



on asbestos in an anaerobic system there was no change in absorption 

 spectrum, indicating that, unlike the ferredoxins (8), the protein cannot 

 be bleached by reduction with a powerful reductant. 



Physical and Chemical Properties . 



By several criteria the protein appeared to be homogeneous. The 

 sedimentation coefficient of the protein dissolved in 50 mM phosphate 

 buffer, pH 7, was measured in the Spinco Model E analytical ultra- 

 centrifuge; the values obtained, extrapolated to infinite dilution, gave 

 a value of S20,w = 1-7. A single sedimenting peak was observed over 

 a concentration range of 0,15 to 0.7 per cent protein. Freshly prepared 

 samples traveled as a single band when subjected to electrophoresis 

 on paper or cellulose acetate foil using 50 mM barbital buffer, pH 8.5, 

 although a small amount of oxidized protein was found after the sample 

 had been stored frozen at -20°C for several months. A single, nearly 

 symmetrical elution curve, with slight tailing, was obtained when 

 purified protein was eluted at constant ionic strength (20 mM Tris 

 plus 40 mM NaCl, pH 8) from an A-25 DEAE-Sephadex column. 



An oxidation- reduction potential determined by titration with buf- 

 fered ferri-ferrocyanide (12) gave the value, E^, pH 7 = +0.35 volt. 

 The ratio of oxidized to reduced iron protein was determined from 

 the absorbancy change at 480 m/^. A one electron change was indicated 

 for the interreaction between the protein and titrant. A commercial 

 grade of cytochrome c became partly reduced by the reduced iron 



