E. M. JOPE 



and the 38-8 per cent solutions, especially in the region of an intense 

 absorption band, so that light losses by reflexion are expected to be 

 a little greater with the stronger solution. This adherence to Beer's 

 law at such high protein concentrations is not so surprising when it 

 is remembered that it represents a concentration of the absorbing 

 haem groups of only about 4 per cent, and that they are probably 

 carefully protected from close proximity to one another by their 

 positioning on the protein carrier molecules and by the sheath of bound 

 water which appears to be carried by the whole molecule 10 . 



It has been found possible to prepare dried uniform films of Hb0 2 , 

 HbCO, and methaemoglobin about 3-10 p. thick which exhibit these 

 visible and Soret bands, and also the farther ultraviolet bands, at the 

 same wavelengths as do these compounds in solution. Quantitative 

 extinction coefficient data are rather more difficult to interpret owing 

 to the different light losses at the quartz/solution and quartz (or air)/ 

 dried protein film interfaces, which cannot be accurately allowed for. 

 With Hb0 2 or HbCO the film must not be dried by reduced pressure 

 unless precautions are taken to keep the 2 or CO pressures above 

 the levels required for keeping the Hb + 2 ^ Hb0 2 and the 

 Hb + CO ^ HbCO equilibria almost completely to the right, other- 

 wise the Hb produced may be gradually oxidised to methaemoglobin 

 and in any case a mixed absorption spectrum will result. It is therefore 

 probable that only in the case of methaemoglobin, and possibly of 

 HbCO, that the spectral absorption of the haem protein can easily be 

 studied in the condition of close-packed molecules with the sheath of 

 bound water removed 10 . This bound water must have been removed 

 in the lyophil dried preparations with Fe contents of 0-31-0-33 per 

 cent n . It is only in such preparations, with haem contents of about 

 10 per cent, that the haem groups of adjacent molecules might be 

 brought into sufficiently close proximity for any interaction leading 

 to changes in spectral absorption to be expected, and comparisons of 

 muscle and red cell haemoglobins under these conditions might yield 

 interesting results. 



With this background of spectral absorption data on haemoglobins 

 under simple known conditions it is possible to proceed profitably to 

 the study of spectral absorption in intact cell structures containing 

 the pigment. There have been reports that the Soret absorption 

 band at 414-5 mjx is suppressed so as to be undetectable in intact red 

 blood cells 12 ' 13 and this has been used 14 as a basis for arguing that 

 Hb0 2 exists in the red cell in a form chemically different from that 

 in solution, combined for instance with stromatin. D. Keilin and 

 E. F. Hartree 15 , while confirming the phenomenon of the absence of 

 the Soret absorption band in the spectral absorption of red cell sus- 



210 



