Further study on myoglobin* 



I. Heterogeneity of human myoglobin 



Some properties of Mb I and Mb II 



A.ROSSI-FANELLI AND E. ANTONINI 



Institute of Biological Chemistry, University of Rome 



In connection with the problem of heterogeneity of crystalHne proteins, we 

 should like to refer to our work on human crystalline myoglobin (Mb). We 

 have demonstrated that at least three components (Mb I, Mb II, Mb III), dif- 

 fering from one another in their electrophoretic behaviour, are present in 

 preparations of human crystalhne Mb.^ The relative percentages of these 

 components are as follows: Mb I, 74%; Mb II, 19%; Mb III, 7%. It has 

 been possible, by means of preparative zone electrophoresis (on paper and 

 on starch geP), to isolate Mb I and Mb II and to study some of their pro- 

 perties. 



As shown in Fig. 1, the isolated components proved to be homogeneous 

 when subjected again to paper electrophoresis. Spectrophotometric analysis 

 of the various derivatives of Mb I and Mb II (Mb, MbOg, MbCO, Mb+) 

 showed that the two components have absorption curves almost identical 

 with each other both in the visible and Soret region and which correspond 

 with those obtained for unfractionated crystalline human Mb. 



A detailed study of the reversible combination with 02^ revealed that 

 Mb I and Mb II are almost identical as regards oxygen equilibrium, either 

 with respect to the shape of the dissociation curve (Figs. 2 and 3) and O2 

 affinity (Fig. 4) at different temperatures. These results seem interesting, 

 particularly as far as the minor component (Mb II) is concerned; this 

 pigment has all the properties of a true myoglobin: absorption spectra, 

 reversible combination with O2, shape of the dissociation curve and affinity 

 for O2, so that we must definitely give up the idea that we are dealing with 

 an impurity or an artefact. 



We must reject also the hypothesis that Mb II is a residue of foetal Mb, 

 since experiments we have carried out demonstrate that foetal Mb cannot 

 be separated by electrophoresis from adult Mb, and that preparations of 

 foetal Mb show the presence of several components which have electro- 

 phoretic characteristics similar to those found in adult Mb. 

 * Aided by a grant from the Rockefeller Foundation. 



