320 ELECTRON TRANSPORT 



protein, but no interaction between purified Chromatium cytochrome 

 552 (Ej^, pH 7 = +0.01 volt) and the iron protein was found to occur. 



The protein contained 2 per cent iron, as found by ashing samples 

 with hot fuming nitric acid and measuring the iron colorimetrically 

 with o-phenanthroline (13). Spectrographic analysis of the iron protein 

 was generously performed by Dr. Bert L. Vallee. No metal other than 

 iron was found in significant amounts. 



In unpublished experiments performed with Dr. Helmut Beinert, 

 EPR spectroscopy of a several month's old preparation of the iron 

 protein showed a broad signal centered at g = 2 and a minor signal at 

 g = 4.3. The signal at g = 4,3 is certainly due to Fe+3 and the signal 

 at g = 2 is in all likelihood also due to Fe+3, The first signal at g = 4.3 

 represents a relatively small amount of high spin Fe+3 and the second 

 signal represents a relatively large amount of Fe+3, probably of the 

 low spin form. The g = 2 signal was extremely sensitive to tempera- 

 ture and broadened rapidly as the sample temperature increased. On 

 addition of ferricyanide the g = 2 signal increased manyfold, indicating 

 that the iron in the starting sample was largely in the reduced state. 

 The signals were abolished by addition of reducing agents such as 

 ferrocyanide and dithionite. 



As noted with other iron- containing proteins such as succinic de- 

 hydrogenase (14) and PPNR (photosynthetic pyridine nucleotide re- 

 ductase) (15), acidification of the iron protein to pH 1 or less caused 

 the liberation of hydrogen sulfide as well as iron, and precipitated a 

 colorless denatured protein. The stoichiometric relation between the 

 H2S evolved and the Fe liberated by such treatment of the iron protein 

 was not determined. 



Amino Acid Analysis and Chemical Constitution. 



Results of amino acid analyses performed according to standard 

 methods (16) by Dr. Dus in the laboratory of Dr. Helen Van Vunakis 

 are summarized in Table 2, where the minimum integral number of 

 amino acid residues is listed. A total of 84 residues were counted, 

 indicating a minimum polypeptide molecular weight of 8842. 



Nonpeptide material in addition to iron and hydrogen sulfide pre- 

 cursor was detected in the protein. Mild acid hydrolysis of the protein 

 in 1 N HCl at 100°C for 4 hours liberated a trace of unidentified 

 carbohydrate, as detected by aniline hydrogen phthalate spray test 

 (20) , plus a yellow- colored material which exhibited blue- white fluores- 

 cence under 254 or 366 m/i irradiation. Similar fluorescent material, 

 which coincided with ninhydrin-stainable material in paper chroma- 

 tograms, was released by enzymatic hydrolysis with trypsin, leucine 

 amino peptidase plus carboxy peptidase, andby pronase. An absorption 

 maximum at 260-270 m/i of this still impure material dissolved in 

 ethanol decreased after the additionof sodium bo rohydride in a manner 

 reminiscent of the behavior of quinones (21). 



