422 M. D. Kamen and R. G. Bartsch 



myoglobin-like spectra are obtained when the pH is brought back to 

 neutrahty. 



Unhke cytochrome c, RHP is rapidly auto-oxidizable. It can be oxidized 

 and reduced reversibly using the reagents commonly employed for this 

 purpose in chemical manipulations of haematin compounds. The alkaline 

 haemochrome form of RHP can also be oxidized and reduced reversibly. 

 However, in the presence of a detergent such as lauryl sulphate, repeated 

 cycles of oxidation and reduction at pH > 11-8 result in progressive loss of 

 haem. This result is not observed at pH 7. The irreversible oxidative 

 degradation in alkah is reminiscent of the behaviour of haemoglobin in the 

 presence of detergents at physiological pH. 



If RHP, while still partially denatured in dilute alkali and in the presence 

 of lauryl sulphate, is incubated with 4-methylimidazole, it forms a typical 

 haemochrome. Denaturation with strong alkali is required before ligands 

 such as cyanide, pyridine, fluoride and azide will bind the central iron atom 

 of RHP. 



The prosthetic group cannot be split by cold acid-acetone, as in myoglobin, 

 haemoglobin, or cytochrome b; reductive cleavage with sodium amalgam, 

 strong acid and heat, or fission with silver salts and acid (Paul, 1951) are 

 required as in cytochrome c. The products obtained by the sodium amalgam 

 procedure (Davenport, 1952) are identical with those derived from cytochrome 

 c. The pyridine and cyanide haemochromes derived after denaturation in 

 strong alkali are identical spectroscopically with those of cytochrome c (Vernon 

 and Kamen, 1954). Hence, it is likely that the prosthetic group of RHP is 

 identical in type and nature of binding with that of the haem in cytochrome c, 

 that is, the haem group is bonded by saturated (thioether) linkages between 

 the vinyl side chains of the haem moiety and cysteine residues of the protein. 



RHP is most peculiar in its response to reagents which normally form 

 reversible addition products with haem. Thus, it does not react at neutral 

 pH with HgS, O2, NO, NaNg, NaCN and 4-methylimidazole, either in the 

 reduced or oxidized form. In this respect, it resembles cytochrome c or 

 cytochrome b. However, it will bind carbon monoxide reversibly in the 

 reduced form to give a typical haemochrome, which is extremely light- 

 sensitive. If the pH is kept below 4, the carbon monoxide complex is very 

 light-stable. 



The chemistry of RHP can be understood on the basis that it is a variant 

 of cytochrome c, capable of reversible denaturation in varying degree, as 

 evidenced by its response to various ligands. The particular variation intro- 

 duced in RHP may be assumed to be a coiling of the peptide chains, so 

 that one or both of the usual basic co-ordination groups of the protein cannot 

 bind to the extraplanar valences of the central iron atom. 



Falk and Nyholm (1957) have presented correlations between electro- 

 chemical potential, spectra, mode of binding and substituents on the 



