212 Discussion 



Lockwood and Rimington (1958), and Mauzerall and Granick (1958) but rate data 

 appear to be lacking. 



EXPERIMENTAL 



In order to test the influence of solubility and aggregation of the complexing agent, 

 two water-soluble porphyrin derivatives were prepared. These substances, porphyrin 

 c and 'porphyrin cytochrome c\ are both closely related to cytochrome c and can 

 thus be regarded as natural products. 



Porphyrin c was obtained in crystalline form by a modification (Neilands and 

 Tuppy, 1959) of the method of Zeile and Meyer (1939). This porphyrin is very soluble 

 in water at all pH values except at the isoelectric point (about pH 4). The a-amino- 

 carboxyl groups in the side chains are probably without steric hindrance to the ap- 

 proach of the ferrous ion ; on the contrary, they may assist in attracting the divalent 

 metal ions to the complexing site. 



'Porphyrin cytochrome c' is the name assigned to cytochrome c minus the iron 

 atom. Besides providing a fully dispersed porphyrin, this derivative offered the 

 additional possibilities of a fifth and sixth co-ordination position for the iron. Por- 

 phyrin cytochrome c was obtained by the following procedure: One micromole 

 (16 mg) of cytochrome c (Sigma Chemical Company) was dissolved in 1-0 ml of pure 

 formic acid containing 10 mg of dry oxalic acid. After the addition of 15mg of 

 platinum (Mohr) catalyst a stream of dry, Oj-free nitrogen was bubbled through the 

 mixture for a period of two hours at room temperature. During this time the solvent 

 evaporated to about one-half of the original volume. A 10 mg batch of ortho- 

 phenanthroline was added, the solution decanted from the catalyst and dialyzed 

 against one liter of distilled water for a period of four hours. The product was 

 chromatographed on a small column of Amberlite XE-64 (Paleus and Neilands, 1950), 

 eluted with 1 M-ammonium acetate and lyophilized. Porphyrin cytochrome c is a 

 water-soluble, intensely fluorescent, purple-coloured protein. The four-banded visible 

 spectrum is sharply delineated thus indicating absence of aggregation in the region 

 of the porphyrin moiety. There was no increase in optical density at 550 m/< in the 

 presence of pyridine and dithionite. Use of the s for porphyrin c at 553 m/f in 

 N HCl gave an apparent molecular weight of 20,000 for the compound. The pre- 

 paration was not checked for the presence of bound water or for initial impurities 

 which may have survived the de-ironing procedure. 



Incorporation of Divalent Ions into Porphyrin c 



The rate of formation of haemin c in the presence of pyridine and sodium dithionite 

 could be followed by direct observation with the Unicam spectrophotometer set 

 at 550 m/ii. All solutes used in the reaction were dissolved in water which had been 

 previously boiled and cooled. A buffer was not employed because of the likelihood 

 of competition of the buffer ions for the iron. The final pH in all experiments was 

 7 to 8 and the temperature was 22° to 25°C. The various ingredients were added 

 to a 1 cm cuvette in the following amounts: 



The optical density increased at a linear rate from an initial value of approximately 

 005 to a terminal figure of about 0-150 after 2 hr. At the end of this period the 

 spectrum of the reaction solution over the range 510 to 560 m/i was that of a typical 

 mesohaemochrome. A control cuvette without iron showed gradual loss of porphyrin 



