The Simultaneous Spectrophotometric 

 Determination of Haemoglobin and 

 Myoglobin in Extracts of Human Muscle 



CHRISTIAN DE DUVE* 



A differential spectrophotometric method is described, by which 

 haemoglobin and myoglobin can be simultaneously determined in 

 extracts of human muscle. The method is based on the spectral 

 differences between the CO-compounds of the two pigments. 

 Readings are taken at 575-7 my., where they have identical extinction- 

 coefficients, at 568 and 583-8 my., where CO-Mgb has the same 

 extinction-coefficients, CO-Hb widely different ones. Subsequent 

 calculations are facilitated by the choice of these particular wave- 

 lengths. Perfect reproducibility of the wave-lengths is an essential 

 condition of accuracy and is ensured by preliminary checking by 

 means of a coloured glass filter. 



The extract used for the determination must be crystal-clear, 

 strongly buffered, and truly representative of the pigment-content of 

 the muscle. A method is briefly described by which these require- 

 ments can be met as completely as possible. Appreciable amounts 

 of unidentified haemin have been found, however, in the tissue 

 residues. By all criteria, they appear to belong to an insoluble 



component. 



The method which is briefly described in this paper was worked out 

 at the suggestion of Professor H. Theorell in order to furnish clinical 

 research workers with a suitable analytical tool, which would make it 

 possible to investigate the myoglobin-content of human heart- and 

 skeletal-muscle under various conditions. The present paper consti- 

 tutes a preliminary report ; detailed publication of the method 

 described has recently been made in Acta Chemica ScandinavicaA 



The main difficulty in the determination of myoglobin is brought 

 about by haemoglobin, which is invariably present in muscle extracts 

 and cannot be removed quantitatively without some loss of myoglobin 

 as well. It was therefore necessary to work out a differential method, 

 taking advantage of the slight spectral differences which exist between 



* Present address : Department of Physiological Chemistry, University of Louvain, 

 Belgium. 



f 2 (1948) 264. 



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