Absorption of Haemoglobins Inside and Outside the Red Blood Cell 



This peculiarity of human foetal haemoglobin persists in the intact 

 red cells, as has been shown by the settled suspension method described 

 above. As judged by this band position, a very specific criterion, the 

 haemoglobin at birth in man is largely of foetal type (shown also by 

 crystal form, this volume, p. 269) and it should be possible to obtain 

 some estimate of the survival of the foetal type of haemoglobin after 

 birth, though no very accurate estimates of the proportion of foetal 

 haemoglobin could be made on such a difference, 1-2 ma. But this 

 small but definite difference between human adult and foetal haemo- 

 globins has the great advantage for cytological research that it is a 

 property observable without disturbing the system chemically, and it 

 should be possible to use the micro-spectroscopic techniques described 

 above to distinguish between adult and foetal haemoglobin within red 

 cell structures, and so learn more of the process of red cell generation 

 in late foetal and early post-natal life. 



These results on the tryptophan band wavelength are summarized 

 in Table II. They were obtained by the moving plate method, and an 

 example of such a spectrogram is illustrated in Figure 15. This is of 

 a native human globin, chosen because it shows clearly the phenyl- 

 alanine band system as well as the tryptophan band. There appears 

 to be much less phenylalanine in foetal than in adult human haemo- 

 globin. Tyrosine is responsible for much absorption at these 

 wavelengths, but does not show prominent fine structure. Figure 16 

 shows a microphotometric record across a spectrogram such as 

 Figure 15, obtained from a settled suspension of adult human red 

 cells : the tryptophan (T) and phenylalanine (P) bands can be seen. 



When globins are prepared from haemoglobins by the HCl-acetone 

 method of M. L. Anson and A. E. Mirsky 35 , the resulting globin 

 shows the tryptophan band at positions varying between about 289 

 and 290 ma. After dialyzing this against phosphate pH 7-4 buffer the 

 material remaining in solution shows this band nearer 290 ma, and 

 is ' native ' according to the criterion of J. L. Crammer and A. 

 Neuberger 36 , who studied spectroscopically the change in pK of the 

 protein tyrosine on denaturation of egg albumin. The product before 

 dialysis contains much material denatured according to this criterion. 

 If the globin soluble at pH 7-4 is recombined with haem the product, 

 although native according to the tyrosine pK. criterion, still gives a 

 tryptophan band at 290 ma. It also gives Soret bands of the Hb0 2 

 and HbCO (after reduction with ferrous tartrate) respectively at 

 406 ma and 416-5 ma instead of the normal 414-5 ma and 420 ma 37 . 

 This recombined haem-globin is not therefore identical with the native 

 haemoglobin from which the globin was prepared, but resembles the 

 second intermediate Hb0 2 denaturation state described above. By 



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