Absorption of Haemoglobins Inside and Outside the Red Blood Cell 



It was clearly desirable to check the validity of the data given by 

 the study of red cell suspensions and layers by extending the study to 

 individual red cells. In the work which has been published up to the 

 present time upon the study of the ultraviolet spectral absorption of 

 individual cell structures by microscopic methods investigation has 

 been concentrated upon absorption intensities at certain wavelengths, 

 the assumption being made that each substance exhibits the same 

 spectral absorption under biological conditions within the cell structure 

 as in simple solution. This was inevitable so long as line sources of 

 light were used to examine the cell structures, giving a limited number 

 of points on the absorption curve, usually insufficient to give absorption 

 peak wavelengths with any accuracy. In order to be able to interpret 

 these intensity data with certainty, however, it is necessary to show 

 for instance that the wavelengths of absorption peaks are the same for 

 substances within the cell structures, approximating to living con- 

 ditions, as they are in simple solutions. This can only be done 

 satisfactorily using light sources giving a continuous spectrum over the 

 whole wavelength range studied. The method described above using 

 settled suspensions of red cells and a continuous source has given 

 valuable information on the Soret band of red cells, necessary for the 

 interpretation of the microspectrophotometric data on intact cells 17 and 

 has indicated a hitherto unsuspected absorption band at 378 mjx which 

 could not have been detected by the usual techniques for examining 

 intact cell structures'-' 3 ' M > 17 > 16 . This suspension technique was 

 further extended to the shorter ultraviolet, using a hydrogen dis- 

 charge tube as a continuous source, with valuable results, showing 

 the absorption band systems of the tryptophan and phenylalanine of 

 the intracellular protein of human red cells, both adult and foetal, 

 to be at the same wavelength position as in simple solution (see below, 

 p. 217 and Table II). 



In order to check the results indicated by the suspension technique 

 on individual red cells, Dr. Holiday and I devised the necessary 

 microspectrographic system for use with a continuous light source. 

 The most convenient method is to illuminate the object in the micro- 

 scope with a continuous spectrum and place the spectral analyzer after 

 the microscope, the image from the latter being focussed on the spec- 

 trograph slit. This is the opposite of the usual arrangement as used 

 by T. Caspersson and others in which the microscope is illuminated 

 from a monochromator, giving a sequence of monochromatic photo- 

 graphs or photocurrent readings. Caspersson's series of monochromatic 

 micrographs or readings are made one after the other over a considerable 

 period of time, whereas by the technique described here these are 

 recorded at all wavelengths simultaneously, thus giving comparable 



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