NONHEME IRON PROTEINS 317 



isolated by the procedure of Newton and Newton (2), implying that the 

 chromatophores are the functional intracellular location of all three 

 proteins. 



Assay Procedure. 



The only assay methods so far used for any of the colored proteins 

 of Chromatium depend on direct spectrophotometric measurements. 

 The iron protein lacks distinctive strong absorption bands and there- 

 fore could not be recognized when mixed with the strongly absorbing 

 cytochromes. Once separated from the cytochromes, the concentra- 

 tion of iron protein was determined by measuring the absorbancy of 

 the 388 m^ absorption band. As a purity index, the ratio of absorbancy 

 at 283 m/U to that at 388 mfi was used. Extinction coefficients of these 

 and other absorption bands of the protein are recorded in Table 1. 



RESULTS 



Spectroscopic Properties . 



Spectra of the oxidized and reduced iron protein in 50 mM phos- 

 phate buffer, pH 7, are compared in Fig. 1. The protein was isolated 

 in the reduced form. To prepare the oxidized form excess potassium 

 ferricyanide was added to the reduced protein dissolved in 50 mM 

 phosphate buffer, pH 7, after which the solution was passed through a 

 G-25 Sephadex column to remove salts from the brown-colored 

 oxidized iron protein. To obtain the reduced spectrum shown in Fig. 1, 

 the oxidized sample was reduced by adding to the solution an approxi- 

 mately 1 mg droplet of pure mercaptoethanol. In the visible region 

 this reduced spectrum was identical to that obtained by reducing the 

 protein with dithionite. In the ultraviolet region the mercaptoethanol 

 did not interfere at wavelengths greater than 250 m^. 



The oxidized-minus- reduced difference spectrum in Fig. 2 was 

 obtained by using oxidized and reduced samples treated like those 

 used for the spectra in Fig. 1. Oxidation of the iron protein resulted 

 in a small decrease in absorption in the UV region, and in an increase 

 in absorption throughout the near UV and visible regions of the 

 spectrum, with the maximum change at 335 m/i and 480 m/i, as shown in 

 Fig. 2. Minor inflections in the absolute spectrum at about 620 and 

 690 m/i and alow, wide absorption band centered at about 1050 m/i were 

 noted with high concentrations of the reduced protein. The spectrum 

 of the reduced protein was unchanged in the presence of carbon monox- 

 ide or 10 mM cyanide. No change in the visible spectrum was ob- 

 served when reduced iron protein was treated with excess dithionite 

 in an anaerobic Thunberg cuvette after the solution had been repeatedly 

 evacuated and then flushed with high purity argon. Similarly, when 

 the protein was treated with hydrogen gas plus 5 per cent palladium 



