Absorption of Haemoglobins Inside and Outside the Red Blood Cell 



pensions, suggested that this effect was purely optical. P. A. Cole 

 and F. S. Brackett 16 had published data showing two points in the 

 Soret band region of the spectral absorption of a single human red 

 cell, which indicated that the Hb0 2 as it existed in the intact red cell 

 was in fact absorbing strongly in this region, and the same is true of 

 the more recent data of B. Thorell 17 . The failure to observe the 

 Soret band in ordinary suspensions of red cells is in fact due to the 

 scattering of light by the cells, and by using different optical systems 

 collecting light over a larger angle E. J. Robinson 18 and D. L. 

 Rubinstein and H. M. Ravikovich 19 have shown the presence of a 

 strong Soret absorption band in suspensions of intact red blood cells. 

 In the region of this strong absorption band the cells tend to behave 

 as semi-opaque particles, scattering the light considerably, and sufficient 

 of this scattered light which has not traversed a haemoglobin path 

 reaches the spectrograph split in the usual spectrographic system to 

 obscure completely the absorption in this region from light which has 

 passed through the red cells. Scattering of light tends to increase 

 with the length of the light path 20 and I have reduced this ratio of 

 scattered light to light transmitted by the cells by allowing the red 

 cells to settle on to a horizontal quartz plate, giving a layer of mean 

 thickness about 1*3 red cells, or of the order of 3 \l. Spectrograms 

 taken by the moving plate method 21 ' 22 with the arrangement shown 

 in Figure 4 show that when the cells required for such a layer are 

 distributed before settling throughout a layer of saline 0-5 cm thick 

 no Soret band absorption can be detected {Figure 5), but that after 

 settling, which takes about \ hour, an intense Soret band is observed 

 (Figure 6). Figure 7 shows a record made from Figure 6 on a Siegbahn 

 recording photometer. Examination of these settled layers under a 

 microscope shows that the red cells settle fairly evenly, and spectro- 

 grams have also been made from blood smears, both wet and dried. 



Spectrograph 

 Slit 1 



Figure 4. System for spectro- 

 graphy of settled red cell 

 suspensions. 



4m 



Thin Layer of -^GsCtfcwte Elf 



III I 



f 5 



Light Source 



_0uar(i 

 Prism 



"L 



Side View 



End View 



The absorption curve of a suspension of human red cells is not 

 quite flat through the Soret band region as illustrated by Keilin and 

 Hartree 15 , but shows a slight rise on the long wave side of the Soret 



211 



