CHRISTIAN DE DUVE 



and ground with dry ice in a mortar until a pinkish homogeneous 

 powder is obtained. This powder is then transferred into a Pyrex glass 

 homogenizer, further ground until thawing is completed and then 

 thoroughly homogenized with 3 ml of a dilute acetate buffer, N/100, 

 pH 4-5. An aliquot of the homogenate is taken for dry weight 

 determination and the remainder weighed and extracted three times 

 with 2 to 3 ml acetate buffer. The three extracts are pooled and their 

 total volume read accurately. After mixing, the pooled extract is 

 examined for clarity and eventually recentrifuged if found not to be 

 crystal-clear. Six ml of the clear supernatant are then pipetted out and 

 alkalinized by the addition of 25 mgm solid Na 2 HP0 4 , 12 H a O per ml 

 of fluid, with a resulting increase in volume of 1-2 per cent. One ml 

 of the alkaline extract is used for pyridine-haemochromogen deter- 

 mination and the remaining 5 for conversion into the CO-compounds 

 and spectrophotometric analysis. It is important to mention that clear 

 extracts can only be prepared by using a slightly acid extracting fluid. 

 If distilled water or alkaline buffers are used, the extracts are turbid 

 and cannot be clarified by centrifuging. Alkalinizing of the extracts 

 before they have been centrifuged to clarity similarly creates unremov- 

 able turbidity. 



Final results are expressed in per cent of dry weight and can be 

 calculated by means of the following formula : 



0/ 1-012 x V x 100 



% Mgb = C Mgb - dw 



in which V is the volume of the extract and C Mgb its Mgb concentration, 

 d.w. the dry weight of the amount of tissue analyzed, which is obtained 

 by multiplying the weight of homogenate extracted by the ratio : dry 

 weight/wet weight of the homogenate. 



Results obtained so far in duplicate and serial determinations on 

 different pieces of the same muscle suggest that the range of error of 

 the method rarely exceeds 10 per cent. 



Appreciable amounts of unextracted haemin have been found, 

 however, in the tissue residues and considerable work has already been 

 devoted to this aspect of the problem. No definite conclusion can be 

 formulated as yet but the evidence available so far does not support 

 the simple explanation that the insoluble haemin residue belongs to 

 unextracted myoglobin. Further results will be communicated by 

 Dr G. Biorck, who has undertaken a systematic study of the myoglobin 

 content of human muscle. 



// lis a pleasure to acknowledge the invaluable help and advice of 

 Professor H. Theorell. . Received August 1948 



REFERENCE 

 1 Theorell, H. and de Duve, Chr. Arch. Biochem. 12 (1947) 113 



128 



