374 



R. W. Henderson and W. A. Rawlinson 



and Neilands, 1950; Margoliash, 1954a, b). Presumably the same material 

 is removed when the iron content is increased by the alkaline salt fractionation 

 of Keilin and Hartree (1945) or the boiling CHCI3 treatment of Tsou (1951a). 

 This colourless fraction was accordingly separated in the Tiselius-type 

 electrophoresis apparatus and found to combine with copper producing a 

 curve of type A, Fig. 2. 



Zinc Precipitation of Myoglobin 



The question arises as to whether the colourless protein fraction is present 

 in vivo. Margoliash (1954a) considered it to be globin present as an artefact 



640 620 600 580 560 540 520 500 480 



Wavelength, m/^ 



Fig. 3. Comparison of characteristic absorption spectra of oxidized and reduced 



cytochrome c samples from ox-heart muscle. Curve A : salt-extracted and Zn- 



treatcd. Curve B: the former after resin treatment. Curve O: the oxidized 



forms of both samples. 



of preparation due to splitting of myoglobin under the conditions of the 

 extraction with trichloroacetic acid (TCA). In the case of neutral extraction 

 procedures some myoglobin was present. In order to examine further this 

 question, cytochrome c was extracted from heart muscle with saline solution. 

 The resulting solution contained the pigments haemoglobin (Hb) and 

 myoglobin (Mb) in addition to cytochrome c. It had been shown by one of 

 us (Rawlinson, 1938) that Hb and globin may be quantitatively precipitated 

 by Zn++ at slightly alkaline pH. Myoglobin was accordingly tested under 

 similar conditions and found to behave in the same manner. Haemoglobin 

 and myoglobin along with much colourless protein material were therefore 

 removed from the saline extract of cytochrome c by this method. Up to 

 this point, the conditions had been such that no splitting of the prosthetic 



