Absorption of Haemoglobins Inside and Outside the Red Blood Cell 



absorption can be clearly seen in the cytoplasm and is absent in the 

 nucleus. From these spectrograms horizontal microphotometric plots 

 can be made of density against wavelength for given parts of the cell 

 structure, or a series of vertical plots can be made giving absorption 

 cross-sections of the cell across the line of the slit at chosen wave- 

 lengths. Figure 12 shows an example of this type of plot, made in 

 this case on a spectrogram of a frog red blood cell using the reflecting 

 microscope and a mercury arc source, the plots being taken at the 

 Hg line wavelengths. Although this is a line spectrum, the lines 

 represent simultaneous exposures of the object. Continuous spectra 

 have to be obtained in the further ultraviolet using a hydrogen arc. In 

 Figure 12 the strong absorption of the cytoplasm and low absorption of 

 the nucleus in the Soret band (405 my) can be seen. This picture 

 changes completely below about 280 m\x, when the nucleoprotein 

 absorption of the nucleus becomes apparent down to 248 m\x. The 

 cytoplasm absorption remains fairly high in this region owing to 

 absorption by the protein part of the haemoglobin. The peaks at the 

 outer edges of the cytoplasm and at the cytoplasm/nucleus interfaces 

 are due to the total reflexion in these regions 17 ' 31 . In an attempt to 

 make the density measurements more quantitative a rotating sector 

 has been incorporated into the system giving in the lower half of the 

 control beam an image of the slit of graded blackening representing 

 object optical densities of 0-3 to 2-3 {Figure 13). This is then scanned 

 on the same photometer record as the cross-section of the cell structure 

 and can be used to derive the cell densities directly {Figure 14). This 

 system has of course rigid requirements in evenness of illumination 

 over the area of field imaged across the spectrograph slit. B. Thorell 17 

 and P. A. Cole and F. S. Brackett 16 use a stepped sector in a similar 

 manner. Figure 14 shows the photometer records from a spectrogram 

 such as Figure 13 of a frog red cell, at two wavelengths, 405 m^, 

 where the haemoglobin of the cytoplasm is absorbing, and at 266 mjx 

 where the nucleoprotein of the nucleus is absorbing. 



Having dealt with the Soret band absorption of haemoglobins in 

 solution and in red cell structures there remains to be treated the 

 further ultraviolet absorption of the protein, due to the aromatic 

 amino acids, tyrosine, trypophan and phenylalanine. First, by a 

 quantitative study of the spectrum of native globin it has been possible 

 to clarify the confusing tryptophan analyses given in the literature 32 : 

 Haemoglobins contain just over 1 per cent tryptophan, and globins 

 prepared from these appear from many analyses (usually by Voisenet 

 technique) to contain more than 2 per cent of tryptophan. By a 

 spectrophotometric analysis by the method of Holiday 33 ' 34 on native 

 human and horse globin preparations it has been possible to show 



215 



