Spectrophotometric Determination of Haemoglobin and Myoglobin 



'(575-7) 



fc '(575'7) 



r --4(568) ~" -4(5 838 ) 



^Hb - 



C-Mgb — ^lot ^Hb 



e Hb(56S) 8 //6(583-8) 



where C = concentration of dry pigment in gm/1, A (x) = density read 

 at x mfx, e w = extinction coefficient at x mji. 



By replacing the extinction-coefficients by their value, one obtains : 



C iot = 1-416 x ^s.t) I 



r C Mgb = C tol — L Hb 



C H b = 2-2123 X (^(568) ~" ^(583-8)) J 



The total pigment concentration (C tot ) was also determined inde- 

 pendently by the reduced pyridine-haemochromogen method, thus 

 providing a check of the value obtained at the isobestic point. 



If 1 ml of the solution to be analyzed is mixed with 3 ml of a solution 

 containing : pyridine 100 ml, N NaOH 30 ml, aq dist ad 300 ml, and 

 if the reading is taken at a wave-length of 557-5 m;x immediately after 

 the addition of an excess of Na 2 S 2 4 , a value of C tot directly com- 

 parable with the one above is obtained from the equation : 



c = ^< 557 ' 5 > 

 ,ot 0-494 



Results obtained in this way can only be accurate if wave-lengths 

 can be reproduced within a fraction of a m\i and this was found not 

 to be the case with the apparatus used. In order to ensure perfect 

 reproducibility of the wave-lengths, the spectrum of a suitably chosen 

 coloured glass filter (Seitz VG 20 1 mm) was determined against air, 

 simultaneously with the two standard spectra, and subsequently used 

 as reference spectrum. Standard readings were made in such a way 

 that each set of three points, corresponding respectively to CO-Hb, 

 CO-Mgb and the filter, were determined without moving the mono- 

 chromator of the apparatus. It was then possible to reproduce sub- 

 sequently any of the wave-lengths used for the standard readings by 

 moving the monochromator to a position where the required extinction- 

 value could be read for the filter. 



The use of this simple device led to a very satisfactory degree of 

 accuracy, the error being less than 1 per cent in most of the trial tests 

 on mixtures of the purified pigments. Good reproducibility was also 

 obtained on muscle extracts, but the preparation of extracts both truly 

 representative of the myoglobin content of the tissue and suitable for 

 spectrophotometric determination turned out to be a problem by itself. 

 After numerous trials, the following method was found most satisfactory : 



Approximately 500 mgm of tissue are cut into small pieces, frozen, 



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